METHODS PAPER

The art of isolating nitrogen-fixing bacteriafrom non-leguminous plants using N-free semi-solid media:a practical guide for microbiologists

Jos Ivo Baldani & Veronica Massena Reis &Sandy Sampaio Videira & Lcia Helena Boddey &Vera Lcia Divan Baldani

Received: 18 December 2013 /Accepted: 24 June 2014 /Published online: 25 July 2014# Springer International Publishing Switzerland 2014

AbstractBackground and aim Nitrogen-fixing bacteria ordiazotrophs have been isolated for many years usingdifferent formulations of N-free semi-solid media. How-ever, the strategies used to isolate them, and the recipesof these media, are scattered through the publishedliterature and in other sources that are more difficult toaccess and which are not always retrievable. Therefore,the aim of this work was to collate the various methodsand recipes, and to provide a comprehensive methodo-logical guide and their use by the scientific communityworking in the field of biological nitrogen fixation(BNF), particularly with non-leguminous plants.

Methods Procedures used for bacterial counting andidentification either from rhizosphere soil or on thesurface of, or within, plant tissues (to access endophyt-ic bacteria) are presented in detail, including colonyand cell morphologies. More importantly, appropriaterecipes available for each N-free semi-solid culture me-dium that are used to count and isolate variousdiazotrophs are presented.Results It is recognized by those working in the field ofBNF with non-legumes that the development of the N-free semi-solid medium has allowed a tremendous ac-cumulation of knowledge on the ecology and physiolo-gy of their associated diazotrophs. At least 20 nitrogen-fixing species have been isolated and identified based onthe enrichment method originally developed byDbereiner, Day and collaborators in the 70s. In spiteof all the advances in molecular techniques used todetect bacteria, in most cases the initial isolation andidentification of these diazotrophs still requires semi-solid media.Conclusions The introduction of the N-free semi-solidmedium opened new opportunities for those working inthe area of BNF with non-legumes not only for eluci-dating the important role played by their associatedmicroorganisms, but also because some of these bacteriathat were isolated using semi-solid media are now beingrecommended as plant growth-promoting inoculants forsugarcane (Saccharum sp.), maize (Zea mays) andwheat (Triticum aestivum) in Brazil and other countries.Further progress in the field could be made by using acombination of culture-independent molecular commu-nity analyses, in situ activity assessments with probe-

Plant Soil (2014) 384:413431DOI 10.1007/s11104-014-2186-6

Responsible Editor: Euan K. James.

J. I. Baldani (*) :V.M. Reis : S. S. Videira : L. H. Boddey :V. L. D. BaldaniEmbrapa Agrobiologia,BR 465 km07, Rio de Janeiro, Seropdica 23891-000, Brazile-mail: ivo.baldani@embrapa.br

V. M. Reise-mail: veronica.massena@embrapa.br

S. S. Videirae-mail: sandyvideira@yahoo.com.br

L. H. Boddeye-mail: boddeylh@hotmail.com

V. L. D. Baldanie-mail: vera.baldani@embrapa.br

S. S. VideiraCentro Universitrio de Volta Redonda (UniFOA),Rio de Janeiro, Volta Redonda CEP 27240-560, Brazil

directed enrichment, and isolation of target strains usingmodified or standard semi-solid media.

Keywords Nitrogen fixation .Media recipes . Isolationprocedure . Bacterial counting . Phenotypiccharacterization

Introduction

The development of the acetylene reduction (AR) tech-nique to evaluate nitrogenase activity in the 1960s(Dilworth 1966; Schllhorn and Burris 1966) and itssubsequent application to investigate BNF associatedwith non-legume crops (Yoshida and Ancajas 1970;Balandreau and Dommergues 1971; Dbereiner et al.1972) generated great interest in the concept that non-legumes may be able to benefit from N inputs from thisprocess. Without doubt, one of the most important pio-neers in the area of BNF associated with non-legumeplants was Dr Johanna Dbereiner, the founder of ourinstitute, now known as Embrapa Agrobiologia. Sinceshe started work at the research institute at Km 47 inSeropdica, Rio de Janeiro, in 1951, she showed a greatinterest in N2-fixing (or diazotrophic) bacteria isolatedfrom rhizosphere soil (Dbereiner 1953) or associatedwith sugarcane (Dbereiner and Ruschel 1958) andother grasses, such as Paspalum notatum (Dbereiner1966). Up until her death in 2000, the team she headeddiscovered and named nine new species of diazotrophsand had developed new techniques to isolate and iden-tify these bacteria (Baldani and Baldani 2005). Evennow, more than 10 years after her passing, all the differ-ent techniques that she and her team developed arescattered across the literature and many of these publi-cations are from the pre-electronic (PDF) period, someare only in Portuguese, and many of them are difficult toaccess. The interest in the bacteria which associate,sometimes endophytically, with non-nodulating plantscont inues unaba ted . For jus t the keywordAzospirillum, the best known nitrogen-fixing andplant growth-promoting genus, the Web of ScienceTM

lists over 3199 publications in all databases since itsdiscovery in 1978 until 2014 (updated March 17,2014). The accumulated literature, especially onAzospirillum, indicates that in addition to BNF, princi-pally in association with sugarcane (Oliveira et al.2006), these bacteria also benefit host plants throughother mechanisms, such as phytohormone and

siderophore production, P-solubilization and biologicalcontrol of phytopathogens (Santi et al. 2013; Compantet al. 2010).

Many researchers require in their work to isolate andidentify these N2-fixing bacteria and, therefore, the ob-jective of this guide is to put together most of theavailable information related to the strategy for isolationand identification of diazotrophs, not only those morefrequently studied by the team of Johanna Dbereiner,but also by other authors both contemporaneous andmore recent who have applied the semi-solid mediumto survey the occurrence of N2-fixing bacteria in theiragricultural systems and climatic conditions.

Development of the semi-solid medium

Until the beginning of the 1970s, reports on N2-fixingbacteria referred only to those bacteria able to growunder atmospheric oxygen concentration (pO2=0.21kPa). However, the key enzyme responsible for thereduction of the N2 gas in the atmosphere, the nitroge-nase complex, is highly sensitive to oxygen and mayinduce irreversible damage to its activity (Dalton andPostgate 1968). Media used to isolate nitrogen-fixingbacteria from the environment were normally preparedin a solid form and only a few diazotrophic bacteriawere described at that time using this traditional ap-proach, such as Beijerinckia fluminensis, Derxiagummosa, D. indica and Azotobacter paspali (Baldaniand Baldani 2005). Also, most of the rhizobia wereisolated using only a solid medium, usually CultureMediumNo. 79 of Fred andWaksman (1928) otherwiseknown as yeast mannitol agar (YMA) (Vincent 1970).The so-called semi-solid medium was initially testedfor different purposes (for details - see Hitchens 1921)and later applied, for example, to measure the reductionof nitrate to nitrite (ZoBell 1932), to detect the oxidationof carbohydrates and distinguish it from fermentation byvarious Gram negative bacteria (Hugh and Leifson1953), and to study bacterial chemotaxis (Adler 1966).The concentration of agar in all these semi-solid mediavaried from 2 to 3 g L1, which was considerably higherthan that defined for the N-free semi-solid medium usedfor isolating N2-fixing bacteria in the 70s byDbereinerand Day (1976). This agar percentage difference be-tween these semi-solid media may be supported by thework of Whittenbury (1963) who developed what hecalled soft agar medium. This author applied the

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medium containing 1.5 g L1 agar to study lactic acidbacteria and concluded that this soft agar provided arange of environments from aerobic to anaerobic andthus permitted the inoculum to develop in that region ofthe medium most suitable for it. This is exactly thebehavior observed for the vibrio-like organisms withvery characteristic corkscrew movements, such as theSpirillum lipoferum described by Beijerinck 1925, andlater reclassified in the newly-created genusAzospirillum (Tarrand et al., 1978). According toDbereiner and Pedrosa (1987), the introduction of theN-free semi-solid medium, such as NFb medium in thecase of Azospirillum (see later for details), was the keywhich allowed for the isolation and identification ofmany nitrogen-fixing bacteria associated with poaceousplants.

The N-free semi-solid medium was originally con-sidered to be a minimal enrichment medium used toimprove the growth of N2-fixing bacteria at the expenseof non- N2-fixing bacteria. No nitrogen source wasadded to the recipe and the pH was adjusted to theoptimal level for each species described based uponhigh acetylene reduction activity (ARA) (Day andDbereiner, 1976). Several media were described withdifferent carbon sources, buffers, mineral concentrationsand the addition, or not, of vitamins. Usually, the semi-solid medium occupies half of the vial volume (10 mL)and allows the inoculation of a single drop of sample(cell, plant tissue or soil suspension) into the center ofthe medium. The consistency of the semi-solid agarmedium is crucial for bacterial growth that is dependenton BNF. Themedium should be neither too solid nor tooliquid, and so the amount of agar added (1.4 to1.8 g L1) needs to be adjusted every time the chemicalcompany providing the agar is changed. In addition, thepH also interferes with the consistency of the medium,and so care should be taken when preparing more acidicmedia, such as JMV and LGIP (see later). To preparesemi-solid media, in each case the agar must be mixedwith the media compounds, melted and distributed intovials before autoclaving. In general terms, the principleof this semi-solid medium is that it allows for growth ofthe bacteria under conditions where their nitrogenaserequires protection from O2-mediated denaturation(Dalton and Postgate 1968) i.e. in early stages of growthwhen only a small number of cells are starting to mul-tiply and they thus use little oxygen. In later stages, thebacterial cells within the culture medium migrate closerto the surface where there is sufficient oxygen pressure

to support aerobic respiration, but not so much that itdamages nitrogenase. With this procedure plant-associated diazotrophic bacterial counts can be madeusing various plant samples, although it was originallydevised only for root samples. From the use of thesemedia the Azospirillum genus rapidly acquired descrip-tions of new species, such asA. amazonense (Magalheset al. 1983), A. halopraeferens (Reinhold et al. 1987)and A. irakense (Khammas et al. 1989).

The original NFb semi-solid medium developed forAzospirillum by Dbereiner and Day (1976) allowed thedevelopment of other media just by replacing the carbonsource, and changing the pH and osmotic concentration,omitting and adding vitamins, salts, amino acids,root/shoot extracts, etc. in order to mimic the environ-ment or plant of interest. Based on these modificationsnew nitrogen-fixing species and even genera were iso-lated and identified in N-free enrichment cultures asshown in Table 1.

Counting and isolating diazotrophic bacteriaassociated with non-legumes

The serial dilution technique is used to count the numberof cells per unit of sample, but also facilitates the pro-cedure to isolate N2-fixing bacteria. Considering that thenumber of bacteria present in the sample is reduced byapplying serial dilution it is recommended that the vialwith the highest dilution showing positive growth (i.e.with pellicle formation) is processed for purification andfurther identification procedures if the interest is toisolate the bacterium that is present in high numbers.This method is also very useful to isolate diazotrophicbacteria that grow under the same conditions but whichare found in lower numbers. The use of semi-solidmedia is considered to be an enrichment method toassess the population present in the sample in a givenharvested time. However, they also allow a survey of thediversity of part of the culturable diazotrophic bacterialpopulation present in the soil or in plant tissues. Figure 1explains in more detail about the steps used for countingand isolating diazotrophic bacteria associated with non-legumes.

Steps for sampling and processing

In the utilization of the Most Probable Number (MPN)method to count and isolate a fraction of culturable

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diazotrophic bacteria, it is assumed that the bacteria arereleased from the soil aggregates and/or plant tissues bythe vortexing/maceration procedure, and that the subse-quent serial dilutions are based on a homogenous sus-pension of individual bacterial cells. It is further as-sumed that single cells of the target organisms can growunder these conditions, and that other bacteria present inthe suspension will not inhibit their growth in the semi-solid medium. Hence, it is apparent that the MPN tech-nique underestimates the population of diazotrophspresent in plant tissues, as was demonstrated by Li andMacRae (1992) and Silva-Froufe et al. (2009), both ofwhom applied the ELISA method to countGluconacetobacter diazotrophicus and Herbaspirillumseropedicae in sugarcane plants growing in the field.Methods applying oligonucleotide probes coupled toepifluorescence microscopy (Stoffels et al., 2001; Wattet al. 2006) or qPCR using strain/species-specificprimers (Pereira 2014) may provide an approximationof the total diazotrophic population associated with the

non-legumes, although more recent tools likemicrobiome studies using 16S rRNA gene sequenceprofiling by next generation sequencing techniques(Turner et al. 2013) or metagenomic studies (Sessitschet al., 2012) probably reveal a more complete picture ofthe N2-fixing community colonizing the rhizosphereand internal tissues of poaceae. A drawback of theselatter methods, of course, is that although they areexcellent for examining bacterial diversity, they do notresult in the isolation of bacterial strains that can then beused for further analyses and/or tested as inoculants.

Nevertheless, the MPN technique has been routinelyused to count and isolate culturable diazotrophic bacte-ria from different parts and tissues of grasses, such asrice (Ferreira et al., 2010), maize (Conceio et al.2009), sugarcane (Reis Junior et al. 2000), foragegrasses (Kirchhof et al. 2001; Brasil et al. 2005;Videira et al. 2012), and has also been applied to fruits,such as banana (Musa sp.) and pineapple (Ananascomosus - Weber et al. 1999), as well as oil palm trees

Table 1 N-free semi-solid media used to isolate non-symbiotic nitrogen-fixing bacteria

Media Bacterial species Carbon source pH References

NFb Azospirillum lipoferum Malic acid 6.8 Dbereiner and Day (1976)

Azospirillum brasilense 6.8 Dbereiner and Day (1976)

Azospirilum irakense 7.0 8.5 Khammas et al. (1989)

Azospirillum doebereinerae Eckert et al. (2001)

Azospirillum melinis 6.5 6.8 Peng et al. (2006)

Azoarcus olearus 6.5 7.3 Chen et al. (2013)

LGI Azospirillum amazonense Sucrose 6.0 6.2 Baldani (1984)

FAM Azospirillum amazonense Sucrose 6.0 Magalhes and Dobereiner (1984)

JNFb Herbaspirillum seropedicae Malic acid 5.8 Baldani et al. (1992)

Herbaspirillum rubrisubalbicans Baldani et al. (1996)

Herbaspirillum frisingense Kirchhof et al. (2001)

JNFb Sphingomonas spp. Malic acid 5.8 Videira et al. (2009)

LGI-P Gluconacetobacter Crystalized cane sugar 5.5 Cavalcante and Dbereiner (1988)

diazotrophicus Reis et al. (1994)

G. johannae Fuentes-Ramrez et al. (2001)G. azotocaptans

JMV Burkholderia kururiensis Mannitol 5.0 5.4 Baldani et al. (2000)

Burkholderia tropica Reis et al. (2004)

Burkhoderia silvatlantica Perin et al. (2006)

Baz Diazotrophic Burkholderia Azelaic acid 5.7 (Estrada-de-los-Santos et al. 2001)

Bac Diazotrophic Burkholderia Azelaic acid+L-citrulline 5.7 (Estrada-de-los-Santos et al. 2001)

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(Elaeis guineensis - Carvalho et al. 2006), and manyother plants in many countries. It can also differentiatethe external from the internal bacterial colonizationby applying a surface disinfection procedure in ad-vance, such as that described by (Baldani et al.1986a), which is detailed below. In addition, theMPN method is easy to apply in any laboratory witha large number of samples, and because it is relativelyinexpensive, the numbers of nitrogen-fixing bacteriasuch as those applied as inoculants can be monitoredquite easily and cheaply. One limitation in monitoringinoculated strains is the difficulty to separate themfrom the endogenous diazotrophic population

associated with the target plants, and in this case theapplication of strain-specific immunological in situdetection methods (James and Olivares 1998;Schloter and Hartmann, 1998; James et al. 2001;Rothballer et al., 2008; Schloter et al. 1995) or quan-titative PCR techniques (Ruppel et al. 2006) aredesirable.

Rhizosphere soil

Rhizosphere soil (i.e. soil around the roots) can either bedirectly inoculated into the N-free semi-solid mediumusing a loopful of soil or a sample (110 g). These

Fig. 1 Diagram showing all thesteps applied to count and isolatefree-living, associative andendophytic diazotrophic bacteria

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samples are normally used in a mixture with 990mL ofsaline solution containing (mg L1) K2HPO4, 100;MgSO4, 50; NaCl, 20; CaCl2.2H2O, 50; FeEDTA,16.4. The addition of Tween 80 (1 g L1) can be appliedto aid bacterial dispersion in soil samples, as well as insamples of bacteria mixed with peat, which is often usedas a carrier for bacterial inoculants. Other methods uti-lize sucrose solution (40 g L1 in water), especially insoil samples collected from the rhizosphere of sugar-richplants like sugarcane and Miscanthus (Eckert et al.2001). The tube containing the 101 diluted solutionshould be capped and shaken vigorously (for ap-proximately 1 h) until the soil is evenly dispersedin the saline solution. The bacterial population inthe rhizosphere, a stable environment that is rich innutrients, is enormous, usually in the range of 106

to 109 viable bacteria per gram of rhizosphere soil(Bulgarelli et al. 2013). Therefore, it is generallysuggested to use serial dilutions down to 106 or109 to count and isolate free-living N2-fixing bac-teria, but these numbers vary with soil moisture, sothe environment conditions at the site where thesamples were collected should always be taken intoconsideration (Oliveira et al. 2004).

Plant samples - roots, stems and leaves

Roots removed from field-grown plants should bewashed in tap water to remove rhizosphere soil. Theyshould then be cut into 10-cm pieces, dried on a papertowel, weighed and then 10 g samples are blended (atapproximately 3,200 rpm) in 90 mL of the above salineor sugar solutions for one to 2 minutes (Fig. 2a). Thesuspensions should then be left to stand for 3060 minafter blending which allows the bacteria to migrate fromthe plant tissue into the suspension. The suspension isthen agitated to homogenize it for 5 to 10min on a rotaryshaker (~150 rpm) and then, 10-fold serial dilutions aremade. Several factors, such as host species, genotypeand plant age, as well as the location of the tissues withinthe plant can all have an impact on the structure of thebacterial communities associated with the plants (vanOverbeek and van Elsas 2008; Bodenhausen et al.2013). Generally, bacterial populations are higheston root surfaces, followed by root internal tissues,and then in the aerial tissues. It is commonly rec-ommended to use serial dilutions down to 107 or109 to count and isolate N2-fixing bacteria fromroots and 105 or 106 from aerial parts.

Fig. 2 First and second steps applied to count and isolatediazotrophic bacteria. a. Sampling and processing plant samples,b. Serial dilution and inoculation into semi-solid media. I. Sam-pling of plants; II. Root-free from soil or substrate; III. Root and

shoot tissues separated; IV. First dilution of samples (101) onsaline solution; V and VI. Serial dilution; VII and VIII. Vortexingsamples before inoculation; IX and X. Inoculation of 0.1 mL intosemi solid media

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Patriquin and Dbereiner (1978) surface disinfectedroots of maize with a reagent active in chlorine knownas Chloramine-T (CH3. C6H4.SO2.N Na Cl.3 H2O) withthe objective of isolating diazotrophic bacteria fromwithin the roots. The isolation of N2-fixing bacteria fromwithin plant tissues using this simple method led to theconcept of endophytic diazotrophs (Dbereiner 1992)based on the definition of Kloepper et al. (1992) whosuggested to replace the term endorhizosphere with thenew term endophytes to refer to microorganisms foundcolonizing inside roots or interior tissues. The termendophyte, which had long been used to refer to (non-mycorrhizal) beneficial fungi living within plants, waslater also used to refer to all bacteria that are able tocolonize the inner tissues of plants without causing anyapparent damage to the host (Hallmann et al. 1997).

A concentration of 10 g L1 of Chloramine-T isrecommended for surface disinfection, and the time ofimmersion in this solution is determined by the age andtype of plant: Usually roots from maize and sorghum atthe flowering stage are immersed for 30 to 60 min,whilst those from rice and wheat can only resist 5 to15 min in the same solution. After surface disinfection,roots should be placed in sterile distilled water for a 1/3of the time that they were in the disinfecting solution.This is repeated for the same time in phosphate buffer(50 mM, pH 7.0) and again in distilled water, with thetotal time amounting to that of the sterilization in Chlo-ramine T. Roots that are not to be disinfected should besoaked in distilled water for the same time as that used in

the sterilization procedure (Baldani et al. 1986a). Theconfirmation of the effectiveness of root surface disin-fection process can be performed using the methodolo-gy described by (Baldani et al. 1986b), where surfacedisinfected roots are capped with paraffin wax on bothends and then submerged in 80 mL of the specific semi-solid medium in large test-tubes. Three days after incu-bation, roots from tubes showing no bacterial growthshould be crushed within the tubes or transferred to newsemi-solid medium, and then the hopefully newly-released microorganisms are allowed to grow, so thatfurther isolation of the endophytic diazotrophic bacteriapresent in the internal tissues can be undertaken.Chloramine-T is effective for roots, but not for aerialtissues. An example of surface disinfection efficiency isshown in Table 2 where zero nitrogenase activity wasobserved when intact roots that were exposed for 1 hourin Chloramine-T and capped with paraffin wax werecompared with sterilized crushed roots. A reduction inthe sterilization time led to positive nitrogenase activityeven for capped roots, thus indicating that the durationof effective surface disinfection is dependent on theplant type, age and tissue, as has been reviewed byHallmann et al. (1997).

In the case of stems e.g. those on sugarcane andelephant grass (Pennisetum purpureum), it is first nec-essary to remove dust and waxes from the surfaces withtap water before proceeding with the disinfection pro-cedure. The stems are sprayed with 70 % ethanol,flamed, and then peeled with a knife to remove theexternal tissue. To ensure that no bacteria from the plantsurface are present in the stems they are dipped into70 % ethanol and flamed again. Leaf samples shouldfirst be washed in tap water and then surface disinfestedwith 70 % ethanol. For counting and isolation of thebacteria in leaf samples they should be treated in thesame manner as described above for soil, roots andstems i.e. 10 g of leaf material are homogenized in90 mL of saline or sucrose solution followed by 10-fold serial dilutions (Videira et al. 2012).

Counting procedures

In most cases, the numbers of non-symbiotic nitrogen-fixing bacteria (free-living, associative and endophytic)are estimated by theMPNmethod (Cochran 1950) usingMcCradys probability tables (Okon et al. 1977). Thisclassic method is based on extinction of the populationusing a serial dilution procedure. Normally, a 10-fold

Table 2 Efficiency of maize root surface disinfection with Chlo-ramineT

Time of exposure toChloramine-T (minutes)

Treatmentsof roots

ARA nmoles C2H4/h/culture roots with8 cm

0 intact 12947

0 crushed 13326

5 intact 846

5 crushed 8631

30 intact 758

30 crushed 1895

60 intact 0

60 crushed 583

- ARA evaluated 42 h after incubation in semi-solid NFb medium

- root tips were immersed in paraffin oil before and afterdisinfection

- Adapted from Baldani (1984)

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serial dilution in saline or sucrose solution is used, andsamples are assumed to differ in predicted numbersbased upon these dilutions. In order to count the cells,0.1 mL of each dilution (generally the highest ones -104 up to 109) are inoculated into vials containing5 mL of each N-free semi-solid medium (Fig. 2b). Thisstep can be performed with 3 or 5 replicates.

Inoculation into semi-solid medium

For most microaerobic diazotrophs the MPN methodusing N-free semi-solid media relies upon the appear-ance of a typical diazotrophic bacterial pellicle in thesubsurface of the medium after incubation for 710 daysat 30C (Fig. 3). Observation of the initial pellicle for-mation is usually possible 23 days after inoculation andgrowth must be then observed every subsequent day assome bacteria can grow very quickly. Counting cangenerally be performed after 57 days growth (Fig. 3).

The characteristic bacterial pellicles in vials with thehighest dilution are transferred to a fresh N-free semi-solid medium, and after confirmation of bacterial

growth, a loopfull of the new pellicle is streaked ontothe corresponding solid semi-specific medium contain-ing a trace amount of yeast extract (around 40 mg L1)to isolate the target bacterium based on the phenotypiccharacteristics of the colonies (Dbereiner 1995)(Fig. 4). A single purified colony is again checkedin the same N-free semi-solid medium and theflasks that originally contained the characteristicpellicle are used for isolation of the bacteria andsubsequent purification on potato agar medium(Baldani et al. 2000) (Fig. 4).

Fig. 3 Inoculation of nitrogen-free semi-solid media and moni-toring the pellicle formed in the media. a. Veil like pellicle formed2 days after inoculation. b. Surface/subsurface pellicle formed7 days after inoculation. The black arrows in figures indicate thecharacteristic pellicle of the diazotrophic bacteria during growth in

different semi-solid media. I. Azospirillum brasilense in NFb 3x,II. Herbaspirillum seropedicae in JNFb, III. Azoarcus olearius inNFb 3x , IV Azospi r i l lum amazonense in LGI , V.Gluconacetobacter diazotrophicus in LGI-P, VI Burkholderiakururiensis in JMV

Fig. 4 Characteristics of colony morphology of severaldiazotrophic bacteria grown on different purification media a.Azospirillum brasilense strain Sp245 on NFb, BMS (Basal Medi-um with Sucrose) and BDA (Potato Dextrose Agar) b. Azoarcusolearius on NFb and BDA c. Azospirillum amazonense strainCBAmC on LGI and BMS d. Herbaspirillum seropedicae strainHRC54 on JNFb, NFb, BMS and BDA; e. Sphingomonas spp.s t r a in BR12195 on JNFb, NFb, LGI and BMS f .Gluconacetobacter diazotrophicus strain PAL5 on LGI-P, Potato-P and BDA; g. Burkholderia kururiensis strain KP23 andB.tropica strain Ppe8 on JMVand on BDA

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Recipes for the N-free semi-solid and solid mediaand description of the bacterial colony types formingon the latter

NFb medium

This medium was initially denominated Fb, after theresearcher Dr. Fabio Pedrosa. After modifications, itwas then denoted NFb (Dobereiner et al. 1976), withthe N meaning new. The basic medium contains(g L1): malic acid, 5.0; K2HPO4, 0.5; MgSO4.7H2O,0.2; NaCl, 0.1; CaCl2. 2H2O, 0.02; micronutrient solu-tion (CuSO4.5H2O, 0.04; ZnSO4.7H2O, 0.12; H3BO3,1.40; Na2MoO4.2H2O, 1.0; MnSO4. H2O, 1.175. Com-plete volume to 1,000 mL with distilled water), 2 mL;bromothymol blue (5 g L1 in 0.2 N KOH), 2 mL;FeEDTA (solution 16.4 g L1), 4 mL; vitamin solution(biotin, 10 mg; pyridoxal-HCl, 20 mg. Dissolve in hot-water bath. Complete to 100 mL by adding distilledwater), 1 mL; KOH, 4.5 g; Distilled water to bring thefinal volume to 1,000 mL and adjust pH to 6.5. Aquantity of 1.6 to 1.80 g agar L1 should be added toprepare the semi-solid medium and 15 g agar L1 for thesolid medium. It is important that the various ingredientsare added in the given sequence to avoid precipitation ofiron or of other salts due to the high pH.

Overall, A. brasilense and A. lipoferum require inoc-ulation into semi-solid NFb medium (Fig. 2, Step 5a).The pellicle should be streaked onto solid NFb, and thenonto BMS (Batata- Malato- Sacarose) agar medium,also known as Potato medium, or on Bacto Dextroseagar (BDA) (Fig. 4a). In BMS the colonies formed areinitially yellowish-white becoming pinkish as they be-come larger and are of wet appearance on BDA (Step5a). Azospirillum doebereinerae (Eckert et al. 2001) isalso commonly isolated with NFb semi-solid mediumafter incubation for 3 to 5 days at 30 C. Further purifi-cation is done on NFb (with yeast extract at 50 mg L1)and half-strength DYGS medium (modified from thatdescribed by Rodrigues Neto et al. 1986) agar plates. OnNFb agar plates with 50 mg yeast extract and triple-strength bromothymol blue, the colonies are 0.5 mm indiameter and appear grey and dull. Colonies grown onmedium with bromothymol blue replaced with congored indicator are scarlet (Rodriguez-Cceres 1982).

The genus Azoarcus (type species, Azoarcus indi-gens) was proposed by Reinhold-Hurek et al. (1993).Some species fix nitrogen and then require microaerobicconditions for growth dependent on N2 (Reinhold-

Hurek and Hurek 2006). The newly-describeddiazotrophic species, Azoarcus olearius (Chen et al.2013) is able to grow in semi-solid NFb medium(Fig. 4b). On NFb agar plates the colonies formed arewhite and wet, while on BDA plates the colonies arelarge, wet and white (Fig. 4b).

LGI medium (Baldani et al. 1984) and FAM Medium(Magalhes and Dbereiner 1984)

The LGI medium, which was developed from LGmedium (Lipman, 1904) with the I derived fromIvo, contains (g L1): sucrose, 5.0; K2HPO4, 0.2;KH2PO4, 0.6; MgSO4.7H2O, 0.2; CaCl2.2H2O, 0.02;Na2MoO4.2H2O, 0.002; bromothymol blue (5 g L

1

in 0.2 N KOH), 5 mL; FeEDTA (solution16.4 g L1), 4 mL; vitamin solution, 1 mL. Completevolume to 1,000 mL with distilled water. Adjust pHto 6.0 to 6.2 with H2SO4 (5 % solution). For semi-solid medium add 1.6 to 1.80 agar L1 and 15 g agarL1 for solid medium.

The FAM medium contains (g L1) sucrose, 5.0;KH2PO4, 0.12; K2HPO4, 0.03; MgSO4.7H2O, 0.2;CaCl 2 , 0 . 02 ; FeEDTA, 0 .066 ; NaCl , 0 .1 ;NaMoO4.2H2O, 0.002; MnSO4, 0.00235; H3BO3,0.0028; CuSO4.5H2O, 0.00008; ZnSO4.7H2O,0.00024; biotin, 0.0001; pyridoxine-HCl, 0.0002 g. Dis-tilled water is added to bring final volume to 1,000 mL.For semi-solid medium add 1.6 g agar L1 and adjust pHto 6.0.

Azospirillum amazonense (Magalhes et al.1983) is best isolated in a semi-solid sucrosebasedmedium, such as LGI (Fig. 4c) or FAM, in thesame way as described above. Sub-surface pelliclesformed after 3 to 5 days incubation at 35 C aretransferred to fresh semi-solid medium and areagain incubated (Fig. 4c). Then the pellicles arestreaked onto solid LGI with 50 mg L1 yeast ex-tract where colonies appear small, whitish, curledwith a firm dense, but not tenacious, consistencyand partially embedded into the agar (Fig. 3b).Individual colonies are transferred into semi-solidLGI medium and then can be streaked onto platesof potato agar (with sucrose as the sole carbonsource) where they will grow as large, flat whitecolonies (5 mm) with raised margins (Fig. 4c).Colony morphology may be altered when differentC-sources are used in the media (for details seeBaldani et al. 2005a).

422 Plant Soil (2014) 384:413431

JNFb medium (Baldani et al. 1992)

Developed by J. Dbereiner from NFb medium(Baldani et al. 1992), the JNFb medium contains(g L1) malic acid, 5.0; K2HPO4, 0.6; KH2PO4, 1.8;MgSO4.7H2O, 0.2; NaCl, 0.1; CaCl2.2H2O, 0.02; Mi-cronutrient solution (above), 2 mL; bromothymol blue(5 g L1 in 0.2 N KOH), 2 mL; FeEDTA (16.4 g L1),4 mL; vitamin solution, 1 mL; KOH, 4.5. Add distilledwater to bring total solution to 1,000 mL. Adjust the pHto 5.8 with KOH. To semi-solid and solid medium add1.8 and 17 g agar L1, respectively.

Herbaspirillum seropedicae, H. rubrisubalbicans andH. frisingense (Baldani et al. 1987; 1996; Kirchhof et al.2001) form a veil-like pellicle in JNFb semi-solid mediumsimilar to that of Azospirillum spp. (Fig. 4d). These bacte-rial pellicles are generally streaked onto JNFb or NFb agarplates containing yeast extract (50 mg L1) where coloniesbecome small and white with a central blue point after1 week incubation (Fig. 4d). This color is more evident inNFbmediumwith three times the normal concentration ofbromothymol blue (denoted NFb 3x) mainly for strains ofH. seropedicae and H. rubrisubalbicans (for details seeBaldani et al. 2005b). Purification on potato medium withsucrose and malate yields small wet raised colonies whichbecome brownish in the center while they remain whiteand wet on BDA (Fig. 4d).

Recently, nitrogen-fixing strains in the genusSphingomonas were isolated using JNFb medium(Videira et al. 2009). The procedure for isolation andpurification is the same as for Herbaspirillum species,but the incubation period is longer than 7 days. Howev-er, in contrast to Herbaspirillum species, white pelliclesare initially formed which later become yellow with lossof the blue color of the JNFb medium (Fig. 4e). Thischaracteristic is considered presumptively to be positivegrowth of Sphingomonas rather thanHerbaspirillum. Inaddition, light green Sphingomonas colonies are formedon JNFB agarmedium, but they appear yellow on potatoagar medium (Fig. 4e).

LGI-P medium (Reis et al. 1994)

Based on the LGI medium (Baldani, 1984), LGI-P wasdeveloped to isolate and count G. diazotrophicus fromsugarcane plants. The letter P stands for the state ofP e r n ambu c o i n B r a z i l , t h e p l a c e wh e r eG. diazotrophicus was first isolated. The composition is(g L1): crystallized cane sugar, 100; K2HPO4, 0.2;

KH2PO4, 0.6; MgSO4.7H2O, 0.2; CaCl2.2H2O, 0.02;Na2MoO4.2H2O, 0.002; bromothymol blue (5 g L

1 in0.2 M KOH), 5 mL; FeCl3.6H2O, 0.01. Add distilledwater to bring total solution to 1,000 mL. Adjust the pHto 5.5 using acetic acid. Add 1.8 g and 17 g agar L1 forsemi-solid and solid medium, respectively.

Gluconacetobacter diazotrophicus (originally namedSaccarobacter nitrocaptans - Cavalcante and Dbereiner,1988), G. azotocaptans and G. johannae (Fuentes-Ramrez et al. 2001) are able to grow in semi-solid LGI-P medium (Fig. 2, Step 5) containing 100 g L1 of crystalsugar (10 %). LGI-P mediumwith 5 mL L1 of sugarcanejuice is called LGI-Pc and is used to recoverG. diazotrophicus from plant samples, especially fromsugarcane (Reis et al. 1994). Seven to ten days afterinoculation of diluted samples into semi-solid LGI-Pc,the vials show orange pellicles and the medium becomescolorless (Fig. 4f). The pellicle is then streaked onto platescontaining solid LGI-P medium amended with 50 mg L1

yeast extract, and incubated for 7 days at 30 C. Coloniesof G. diazotrophicus are small and orange (Fig. 4f). Thecolonies ofG. johannae are yellow-orange, very irregular,smooth and flat after 5 d growth. The G. azotocaptanscolonies are orange but form round, mucoid, smooth andconvex colonies with translucent margins. The purifica-tion step is made by streaking colonies onto Potato-Pmedium, where colonies ofG. diazotrophicus are initiallymoist and clear, changing to chocolate brown 7 to 10 daysafter incubation at 30 C (Fig. 4f).

JMV (Baldani, 1996), BAz and BAc media(Estrada-de-los-Santos et al. 2001)

The JMV medium contains (g L1) mannitol, 5.0;K2HPO4, 0.6; KH2PO4, 1.8; MgSO4.7H2O, 0.2; NaCl,0.1; CaCl2.2H2O, 0.2; bromothymol blue (5 g L

1 in0.2 N KOH), 2 mL; FeEDTA (16.4 g L1), 4 mL; Mi-cronutrient solution (see above), 2 mL; vitamin solution,1 mL. Add distilled water to bring total solution to 1,000mL. Adjust the pH to 5.0 - 5.4 with KOH. To semi-solidand solid medium add 1.8 and 25 g agar L 1 respectively.Yeast extract (0.1 g) can be added in semi-solid mediumto stimulate growth of pure culture when evaluated inlaboratory conditions.

BAz medium has the following composition (g L1):azelaic acid, 2.0; K2HPO4, 0.4; KH2PO4, 0.4; MgSO47H2O, 0.2; CaCl2, 0.02; Na2MoO4H2O, 0.002; FeCl3,0.01; bromothymol blue, 0.075; and agar, 2.3. Adjust thepH to 5.7 with KOH. Add distilled water to bring total

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solution to 1,000 mL. Vials containing 5 ml of BAzmedium are autoclaved at 121 C for 20 min, and filter-sterilized cycloheximide (200 g/tube) is then added.

The BAc medium contains (g L1): azelaic acid, 2.0;L-citrulline, 0.2; K2HPO4, 0.4; KH2PO4, 0.4 andMgSO47H2O, 0.2. Adjust the pH to 5.7 with KOHand the medium is sterilized at 121 C for 20 min priorto the addition of filter-sterilized (pore size, 0.22 m)citrulline as the sole nitrogen source. Add distilled waterto bring total volume to 1,000 mL.

Nitrogen fixing species, such as the BrazilianBurkholderia kururiensis strains (Baldani and Baldani2005) as well as the species B. tropica (Reis et al. 2004),B. silvatlantica (Perin et al. 2006) and B. unamae (Cabal-lero-Mellado et al. 2004) are easily isolated applying theJMV medium containing mannitol as a carbon source.However, the BAz and BAcmedia were also used byReiset al. (1994) and Caballero-Mellado et al. (2004) to isolateand cultivate theseBurkholderia species. Other recipes arealso described by (Estrada-de-los-Santos et al. 2001) toisolate new species of Burkholderia from plant samples.Bacteria of this genus are very versatile and can also growin LGI and LGI-P medium. The serial dilutions are inoc-ulated into the semi-solid medium and incubated 4 to7 days at 32 C. On the fourth day thick pellicles willbegin to form (Fig. 4g). Once these pellicles have migrat-ed to the surface of the medium, they are streaked ontoplates of JMV solid medium amended with 60 mg L1

yeast extract and again incubated for 4 to 7 days at 32 C.Colonies are brownish in the center and light browntowards the edge (Fig. 4g). The colonies are light-brownin the potatomedium andwhite andwet in BDA (Fig. 4g).It should also be noted that several new diazotrophicBurkholderia species have been described that are symbi-otic with legumes, most notably ofMimosa spp. in Brazil.Some of these, such as B. phymatum and B. tuberum havealso been shown using semi-solid JMV medium plusyeast extract to fix N2 ex planta (Elliott et al. 2007), whichis a highly unusual feature for legume-nodulating bacteria.

Additional media for enrichment and isolationof N2-fixing bacteria

OAB semi-solid nitrogen-free medium (Okon et al.1977)

The original NFb medium of Dbereiner and Day(1976) was modified by Okon et al. (1977) to provide

increased buffer capacity and micronutrient elementsto isolate and count Azospirillum lipoferum frompure culture and from inoculated maize plants. Themedium contains the following (per liter of distilledwater): K2HPO4, 6.0 g; KH2PO4, 4.0 g (mixed in0.1 the final volume and autoclaved separately fromthe other medium constituents; the phosphate solu-tion is later mixed with the cold medium);MgSO4.7H20, 0.2 g; NaCl, 0.1 g; CaCl2, 0.02 g;DL-malic acid, 5.0 g; NaOH, 3.0 g; FeCl3, 10.0 mg;NaMoO4.2H20, 2.0 mg; MnSO4, 2.1 mg; H3BO3,2.8 mg; CuSO4.5H2O, 0.04 mg and ZnS04.7H20,0.24 mg. The final pH is adjusted to 6.8. For growthon N2 under microaerophilic conditions, no NH4Clor yeast extract are added to the medium, but 0.5 gof agar are added per liter.

Combined carbon or Rennie semi-solid medium(Rennie 1981)

The combined carbon or Rennie semi-solid medium(RM) is prepared from solutions A and B. Solution Aconsists of 0.8 g of K2HPO4, 0.2 g of KH2PO4, 0.1 g ofNaCl, 28 mg of Na2FeEDTA, 25mg of Na2MoO4.2H2O,100 mg of yeast extract (Difco), 5.0 g of mannitol, 5.0 gof sucrose, 0.5 ml of 60 % (vol/vol) sodium lactate and900 mL of distilled water (the final pH of solution A isadjusted to 7.0 before autoclaving). Solution B consists of0.2 g of MgSO4.7H2O, 0.06 g of CaCl2.2H2O, and100 mL of distilled water. The solutions are autoclavedseparately and mixed after cooling. Filter-sterilized biotinand para-aminobenzoic acid (100 l each) are added atfinal concentrations of 5 and 10g L1, respectively. Add2.0 g and 15 g agar L1 for semi-solid and solid medium,respectively.

M medium (Xie and Yokota 2005)

The nitrogen-fixing bacterium Azospirillum oryzae (Xieand Yokota 2005) was isolated using medium M whichcontains (g L1): sodiummalate, 5.0; CaCl2.2H2O, 0.02;MgSO4.7H2O, 0.2; K2HPO4, 0.1; KH2PO4, 0.4; NaCl,0.1; FeCl3, 0.010; Na2MoO4.2H2O, 0.002; yeast extract,0.1 and biotin (2 g). Adjust the pH to 6.8 with KOHand add distilled water to bring the total volume to 1,000mL. To semi-solid medium add 2.0 g agar L 1.Azospirillum canadense (Mehnaz et al. 2007) can alsobe isolated using M medium, except that biotin is omit-ted and pH is adjusted to 7.2 to 7.4.

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Semi-solid synthetic malate (SSM) medium (Reinholdet al. 1986, Reinhold et al. 1987)

The semi-solid synthetic malate (SSM) medium wasadapted for isolating diazotrophs from the high saltconcentrations commonly found in soils where Kallargrass (Leptochloa fusca) is found. SSM was used foren r i chmen t and i so l a t i on o f Azosp i r i l l umhalopraeferens and also of Azoarcus spp. (Reinhold-Hurek et al. 1993). The medium has the followingcomposition (g L1): malic acid, 5.0; KOH, 4.8; NaCl,1.2; Na2SO4, 2.4; NaHCO3, 0.5; CaC12, 00.22; MgSO4- 7H2O, 0.25; K2SO4, 0.17; Na2CO3, 0.09; Fe (II1)(EDTA), 0.077; K2HPO4, 0.13; biotin, 0.0001;MnC12.4H2O, 0.0002; H3BO3, 0.0002; ZnC12,0.00015; CuC12 - 2H2O, 0.00002; Na2MoO4 2H2O,0.002 and 2 g agar. For solid medium add agar (8 g)and yeast extract (0.1 to 0.2 g). The final pH of themedium is adjusted to 8.5, and distilled water is added tobring the total volume to 1 liter.

Media used for purification of diazotrophic bacteriaand stock solutions

BMS or Potato agar medium

BMS (Potato malate sucrose) medium has the fol-lowing composition (g L1): potatoes peeled and sliced,200; DL-malic acid, 2.5; KOH, 2.0; crystalized canesugar, 2.5; vitamin solution (see above), 1.0 mL; Micro-nutrient solution (see above), 2 mL; bromothymol blue(5 g L1 in 0.2 N KOH), 2 drops; agar, 15.0. Thepotatoes are placed in a gauze bag, boiled in 0.5 l ofwater for 30 min, and then filtered through cotton,saving the filtrate. The malic acid is dissolved in50 mL of water and the bromothymol blue added.KOH is added until the malic solution is green(pH 6.8-7.0). This solution, together with the crystalizedcane sugar, vitamins and agar, is added to the potatofiltrate. The final volume is made up to 1 L with distilledwater. The medium is boiled to dissolve the agar andthen sterilized by autoclaving.

Potato-P agar medium (Dbereiner 1995)

This is similar to the BMS medium, but the concentra-tion of crystallized cane sugar is increased to 100 g L1,malic acid is omitted and the pH is adjusted to 5.5. This

medium is specifically applied to the final purification/characterization of the N2-fixing Gluconacetobacterdiazotrophicus.

Rojo Congo (RC) agar medium (Rodriguez-Cceres1982)

The RC agar medium, claimed to improve the isolation/identification of Azospirillum spp. (colonies show ascarlet color), contains (g L1): DL-malic acid, 5;K2HPO4, 0.5; MgSO4.7H20, 0.2; NaCl, 0.1; yeast ex-tract, 0.5; FeCl3.6H20, 0.015; KOH, 4.8; and agar, 20.The pH is adjusted to 7.0 with 0.1 M KOH. A total of15 mL of a 1:400 aqueous solution of Congo red(autoclaved separately) is aseptically added to each literof the melted medium just before use.

DYGSmedium Rodrigues Neto et al. (1986) modified

DYGS medium was developed for the enrichment andisolation of Xanthomonas spp. by Rodrigues Neto et al.(1986). Later, the composition was modified and usedfor the purification of diazotrophic bacteria. The com-position is (g L1): glucose, 2.0; malic acid 2.0; peptone,1.5; yeast extract, 2.0; K2HPO4, 0,5; MgSO4.7H2O, 0.5;glutamic acid, 1.5; complete with distilled water up to1,000 mL. The optimum pH is 6.0 for Herbaspirillumspp. and Gluconacetobacter spp. and 6.8 forAzospirillum spp. Usually, Gluconacetobacter spp.grow well using DYGS medium without malic acid.

Advantages and limitations of the N-free semi-solidmedia

It is recognized by those working in the field of nitrogenfixation with non-legumes that the development of the N-free semi-solid medium allowed a tremendous accumula-tion of knowledge on the ecology and physiology ofdiazotrophic bacteria. At least 20 N2-fixing species havebeen isolated and identified based on the enrichmentmethod originally developed by Dbereiner and Day(1976). Because of the simplicity of the method, it hasalso been used to estimate the population of diazotrophicbacteria associated with rhizosphere soil and plant tissues,although it almost certainly underestimates diazotrophicbacterial populations as discussed previously. Dependingon the type of study, a specific culturable diazotrophicspecies population may be counted, especially when

Plant Soil (2014) 384:413431 425

combining media with antibiotics to target specificstrains, as used in a study carried out by (Baldaniet al. 1986b, Baldani et al. 1987) to monitor theestablishment of inoculated Azospirillum brasilensestrains Sp245str and Sp 107str in wheat andA. lipoferum strain Sp S82str in sorghum. In addition,it is possible to perform ARAs directly on the bacteriagrowing in the semi-solid medium or even to determinetheir N2 fixation ability by measuring the

15N incorpo-rated into their cells after exposure of the culture to15N2 gas (Baldani et al. 1992). These procedures em-phasize the importance of semi-solid media for widerphysiological characterization studies of diazotrophicbacteria in programs designed to select strains for plantinoculation or to exploit the diversity of N2-fixingbacteria associated with various non-legumes.

Despite the aforementioned advantages of the semi-solid medium, it presents some limitations that need tobe taken into account: a) The method allows the identi-fication of only a fraction of the diazotrophic bacterialpopulation present in the plant tissues or in soils; b)Although they were designed to be specific for particu-lar diazotrophic genera, it is possible when using thesemedia to isolate other diazotrophic bacteria that use thesame carbon sources or which tolerate the same pH levelas for example the nitrogen-fixing species belonging tothe genera Stenotrophomonas, Pantoea, Enterobacter,etc. that were isolated from sugarcane using the LGI,LGI-P and JNFb media (Taul et al. 2012); c) At least100 bacterial cells are necessary to initiate growth andthe actual number of cells depends on the medium usedand, d) Non-N2-fixing bacteria (scavengers) can growtogether with the diazotrophs during the incubationperiod (pellicle formation) due to the fixed nitrogenreleased by the diazotrophic bacteria, and these mayinterfere with the process of isolation and purificationof the target (diazotrophic) bacteria. In order to reducethis latter problem, Dbereiner (1988) recommendedthat the pellicle should be transferred to another semi-solid medium before initiating the isolation process withsolid medium. It is important to verify the pellicle for-mation, as many other (non-diazotrophic) bacteria canutilize (i.e. scavenge) small amounts of fixed N presentin the medium. The 10 commandments listed byDbereiner (1988) indicate the procedures, includingthe necessity for the demonstration of least a minimaldegree of nitrogenase (acetylene reduction) activity, tobe followed when seeking success in isolating N2-fixingbacteria from non-legumes.

Future Perspectives

In conclusion, the introduction of the N-free semi-solidmedium opened new opportunities for those working inthe area of BNF with non-legumes, not only for theimportant role played by microorganisms associatedwith cereals and energy crops but also because someof these diazotrophs, most notably Azospirillum spp.,are being recommended as inoculants for sugarcane,maize and wheat in Brazil and in other countries, suchas Argentina, Mexico, Colombia, Egypt and South Af-rica, among others. Azospirillum spp. have also beenused as co-inoculant plant growth-promoting bacteria tobe applied with soybean rhizobia in Argentina andSouth Africa for many years. The recommended meth-odologies applied to count these bacteria in formulationsinclude recipes of semi-solid and solid media, thusillustrating their importance in the general field of BNF.

Molecular approaches, such as qPCR can be appliedto detect with high efficiency a target N2-fixing speciesor even a diazotrophic strain colonizing soil or planttissues (Quecine et al. 2012). nifH cDNA clone librarieshave also been applied to detect the functionaldiazotrophic bacterial community associated with manypoaceous plants (Fischer et al. 2012; Videira et al. 2013;Demba-Diallo et al. 2008; Ando et al. 2005). To facili-tate the isolation of these in silico identified diazotrophicbacteria, traditional microbiological methods (via isola-tion using semi-solid media) can be coupled to a molec-ular probe-directed approach that may be used to predictthe culturable diazotrophic diversity present in the planttissues (Hartmann et al. 2006). Several molecular probeshave already been designed (Stoffels et al., 2001;Rothballer et al. 2006) and applied to monitor N2-fixingproteobacteria associated with non-legumes (Schloteret al. 1995; Oliveira et al. 2009).

The majority of the diazotrophic bacteria associatedwith non-legumes have been obtained using the recipeslisted earlier. However, considering the published liter-ature on this subject the possibilities to isolate newspecies and even genera are very high using novelsemi-solid media. For example, a simple modificationin the NFbmedium by the addition of 3%NaCl allowedthe isolation from the Brazilian coral speciesMussismilia hispida of nineteenVibrio strains belongingto species V. harveyi, V. alginolyticus, V. campbellii, andV. parahaemolyticus. Many of these vibrios were capa-ble of growing six consecutive times in N-free mediumand each time showed strong nitrogenase activity

426 Plant Soil (2014) 384:413431

(Chimetto et al., 2008). A similar strategy was used byJha et al. (2012). Simply by adding 4%NaCl to the NFbsemi-solid medium enabled them to isolate manydiazotrophic Gram positive (Brachybacteriumsaurashtrenese, Brevibacterium sp. and Zhihengliuellasp.) and Gram negative (Haererehalobacter sp.,Halomonas sp. and Mezorhizobium sp.) diazotrophicbacteria from the halophyte Salicornia brachiata grow-ing in India. The modification of the semi-solid LGImedium by addition of chlorogenic acid, caffeic acid,substituting glucose for sucrose and adjusting pH to 6.8,allowed the isolation of an endophytic N2-fixing Kleb-siella oxytoca strain from sweet potato (Ipomoeabatatas) stems growing in Japan (Adachi et al. 2002).

Other strategies to isolate new species, mainly thoseidentified in silico but not yet cultivated, should becontinuously investigated. For example, the use of asmall amount of filtered root or stem extract increasesthe diversity of N2-fixing bacteria isolated from targetnon-legumes, as exemplified by Elbeltagy et al. (2001)who added rice shoot extracts to the semi-solid RMmedium (Rennie 1981) and this allowed for the isolationfrom rice plants of several diazotrophic bacteria phylo-genetically related toHerbaspirillum, Ideonella, Entero-bacter, and Azospirillum. In this case, however, careshould be taken into considering the presence of thenutrient N in the plant tissues used to supplement themedia, as it may stimulate the growth of non-N2-fixingbacteria. The use of a semi-solid medium containingmore than one carbon source, mainly those related toexudates released by the host plant, has also allowed forthe isolation of a greater diversity of N2-fixing bacteria.For example, the use of semi-solid NFb COG medium(malic acid replaced by citric acid, oxalic acid andglucose - ratio 1:1:3 g L-l), pH 5.5, allowed for theisolation of many diazotrophs from wetland rice plants,including Azospirillum, Herbaspirillum and an uniden-tified species named Bacteria E (Oliveira 1992),which was later characterized as belonging to the genusBurkholderia (Hartmann et al. 1995).

Therefore, the options for the creation of new semi-solid media to survey new bacterial species and toexploit the diversity of diazotrophic bacteria are vastand depend only on the input of the younger researchscientists starting out in the field of non-legume BNFthat has just reached its half century. One example is thesuccessful strategy applied by Rouws et al. (2013) toisolate many endophytic Bradyrhizobium strains fromsugarcane plants based on culture-independent

molecular information that indicated the presence ofbradyrhizobia colonizing the plant (Fischer et al.2012; Burbano et al. 2011). Another example is theapproach carried out by (Lonhienne et al. 2014) thatfirst identified bacteria markedly enriched in the rhizo-sphere of sugarcane plants based on culture-independentbacterial community assessment using 16S rDNAamplicon sequencing to guide the isolation of N2-fixingstrains, such as the new species Burkholderia australisthat was derived from their study.

Finally, in order to search for, isolate and confirm thatparticular diazotrophs have an associative/endophyticability to colonize non-legumes and/or that they performefficiently when applied as a PGPR in agriculture, werecommend a combination of sophisticated culture-independent molecular approaches (including high res-olution microscopy) together with the application ofsimple microbiological methods that utilize standardand/or modified nitrogen-free semi-solid media. Indeed,the power of such a combined traditional and molec-ular approach guarantees that it will most certainly berequired for many years to come.

Acknowledgments The authors thank Embrapa Agrobiologia,the CNPq/INCT-FBN and FAPERJ for financial support andCNPq for the fellowship of the researchers of EmbrapaAgrobiologia. Thanks also to our colleague Robert M. Boddeyfor his encouragement to write this guide and reading the manu-script. The authors thank the laboratory analist Fernanda Douradofor the bacterial photographs.

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The...AbstractAbstractAbstractAbstractAbstractIntroductionDevelopment of the semi-solid mediumCounting and isolating diazotrophic bacteria associated with non-legumesSteps for sampling and processingRhizosphere soilPlant samples - roots, stems and leavesCounting proceduresInoculation into semi-solid medium

Recipes for the N-free semi-solid and solid media and description of the bacterial colony types forming on the latterNFb mediumLGI medium (Baldani etal. 1984) and FAM Medium (Magalhes and Dbereiner 1984)JNFb medium (Baldani etal. 1992)LGI-P medium (Reis etal. 1994)JMV (Baldani, 1996), BAz and BAc media (Estrada-de-los-Santos etal. 2001)

Additional media for enrichment and isolation of N2-fixing bacteriaOAB semi-solid nitrogen-free medium (Okon etal. 1977)Combined carbon or Rennie semi-solid medium (Rennie 1981)M medium (Xie and Yokota 2005)Semi-solid synthetic malate (SSM) medium (Reinhold etal. 1986, Reinhold etal. 1987)

Media used for purification of diazotrophic bacteria and stock solutionsBMS or Potato agar mediumPotato-P agar medium (Dbereiner 1995)Rojo Congo (RC) agar medium (Rodriguez-Cceres 1982)DYGS medium Rodrigues Neto etal. (1986) modified

Advantages and limitations of the N-free semi-solid mediaFuture PerspectivesReferences