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Review article

Phosphate-solubilizing bacteria as inoculantsfor agriculture: use of updated molecular

techniques in their study

Jos Mariano IGUALa*, Angel VALVERDEa, Emilio CERVANTESa, Encarna VELZQUEZb

a Instituto de Recursos Naturales y Agrobiologa, Consejo Superior de Investigaciones Cientficas,

Apartado 257, 37071 Salamanca, Spainb Departamento de Microbiologa y Gentica, Edificio Departamental, Universidad de Salamanca, 37007 Salamanca, Spain

(Received 18 December 2000; revised 3 July 2001; accepted 30 August 2001)

phosphate-solubilizing bacteria/ 16S rRNA/ LMW RNA/ TP-RAPD / rep-PCR/ DNA probes

RsumLes bactries qui solubilisent les phosphates comme inoculants en agriculture : utilisation de techniques molcu-laires rcentes pour leur tude. Lutilisation de bactries qui solubilisent les phosphates comme inoculants augmente en mmetemps labsorption de cet lment par la plante ainsi que le rendement des cultures. Cet article contient une br ve revue des tech-niques rcentes de biologie molculaire utilises pour caractriser ces bactries (squenage du 16S RNA, profils de LMW RNA, etempreintes par TP-RAPD et rep-PCR). On prsente aussi une mthode disolation de souches efficientes pour la solubilisation duphosphate et une mthode pour obtenir des sondes d'ADN spcifiques des souches. Il sagit de mthodes rapides qui, en gnral,nont pas besoin dun quipement sophistiqu.

bactrie dissolvant les phosphates / 16S rARN / LMW RNA / TP-RAPD / rep-PCR / sonde ADN

1. INTRODUCTION

Phosphorus is one of the major plant nutrients limit-

ing plant growth. Most agricultural soils contain largereserves of P, a considerable part of which has accumu-lated as a consequence of regular applications of chemi-cal fertilizers. However, a large proportion of soluble

inorganic phosphate added to soil is rapidly fixed asinsoluble forms soon after application and becomesunavailable to plants [51]. Phosphorus fixation and pre-

cipitation in soil is generally highly dependent on pHand soil type. In acid soils, free oxides and hydroxides ofAl and Fe fix P, while in alkaline soils it is fixed by Ca,causing a low efficiency of soluble P fertilizers. The

Agronomie 21 (2001) 561568 561 INRA, EDP Sciences, 2001

Communicated by Jean-Jacques Drevon (Montpellier, France)

* Correspondence and reprintsigual@gugu.usal.es

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calcium superphosphate fertilizer, which contains about15% of phosphorus pentoxide, normally loses its avail-able P proportion when it comes in contact with soilminerals containing calcium carbonates [31].

A substantial number of bacterial species, mostlythose associated with the plant rhizosphere, may exert abeneficial effect upon plant growth [17]. This group ofbacteria has been termed plant growth promoting rhi-zobacteriaor PGPR [28] and, among them, some phos-phate-solubilizing bacteria (PSB) are already used ascommercial biofertilizers for agricultural improvement[51, 58].

Although plant growth promoting rhizobacteria occurin soil, usually their numbers are not high enough tocompete with other bacteria commonly established in therhizosphere. Therefore, for agronomic utility, inoculationof plants by target microorganisms at a much higher con-

centration than those normally found in soil is necessaryto take advantage of their beneficial properties for plantyield enhancement. Root colonization is an importanttrait of rhizobacteria and can be strain-specific. Thus, toscreen natural resources for better strains, reliable identi-fication and detection methods are needed.

This paper presents, in a concise way, the rationale forplant inoculation with PSB, methods for screening andselecting elite strains of PSB, some updated moleculartechniques to identify them, and a practical approach toobtaining DNA probes as useful tools for studying theirbehavior in soil.

2. THE RATIONALE FOR PLANTINOCULATION WITH PSB

Ectorhizospheric strains from pseudomonads andbacilli, and endosymbiotic bacteria from rhizobia havebeen described as effective phosphate solubilizers. Theproduction of organic acids is considered as the principalmechanism for mineral phosphate solubilization in bac-teria. This assumption has been corroborated by cloningof two genes involved in gluconic acid production: PQQsynthase [18, 32, 52] and gabY[3] genes. Gluconic acidis the principal organic acid produced by Pseudomonassp. [26], Erwinia herbicola [32], Pseudomonas cepacia[19] and Burkholderia cepacia [51]. Rhizobium legumi-nosarum [24], Rhizobium meliloti [22] and Bacillus fir-mus [4] produce noticeable amounts of 2-ketogluconicacid. Other organic acids, such as lactic, isovaleric,isobutyric, acetic, glycolic, oxalic, malonic and succinicacids are also generated by different phosphate-solubiliz-ing bacteria [51].

Beneficial effects of inoculation with phosphorus-sol-ubilizing microorganisms to many crop plants have been

described by numerous authors [11, 41, 49, 51, 53, 58,59]. Rhizobia are, perhaps, the most promising group ofPSB on account of their ability to fix nitrogen symbioti-cally with legumes and the capacity of some strains for

solubilizing insoluble inorganic phosphate compounds[23, 24]. Several publications have demonstrated thatphosphate-solubilizing strains of Rh izo bi um an dBradyrhizobium increase growth and P content of nonleguminous as well leguminous plants [2, 6, 7, 43].

An alternative approach for the use of phosphate-solu-bilizing bacteria as microbial inoculants is the use ofmixed cultures or co-inoculation with other microorgan-isms. In this regard, some results suggest a synergisticinteraction between vesicular arbuscular mycorrhizae(VAM) and PSB which allows for better utilization ofpoorly soluble P sources [14, 16, 27, 44, 47, 60, 61].Similarly, plant growth can be increased by dual inocula-

tion with PSB and Azospirillum [1, 5] or Azotobacter[29, 38].

3. ISOLATION AND SELECTION OF PSBTO BE USED AS INOCULANTS

Phosphate-solubilizing bacteria are isolated from rhi-zospheric samples by plating serial dilutions of rhizos-pheric soil extracts in Pikovskayas solid medium [45].That medium contains insoluble tri- or bi-calcium phos-phate, allowing the detection of phosphate-solubilizermicroorganisms by the formation of halosaround their

colonies. The addition of bromophenol blue, which pro-duces yellow coloured halos around the colonies inresponse to the pH drop by the release of organic acids,or proton release in exchange for cation uptake, gener-ates more reproducible results than with the simple halomethod [21]. Although phosphate-solubilizing capabilityremains stable in most isolates, some strains show insta-bility of this trait after several cycles of inoculation [24,26]. Thus, the persistence of phosphate-solubilizingcapacity after five or more subcultures should be the firstcriterion in selecting bacterial strains as microbial inocu-lants. Last of all, the identification of the most efficientphosphate solubilizers in vitro has to be done by quanti-fying their phosphate-solubilizing capacities in liquid

cultures since, in some cases, there have been contradic-tory results between plate halo detection and phosphatesolubilization in liquid cultures [51]. In this regard, anovel defined microbiological growth medium (NBRIP),which demonstrated about 3-fold higher efficiency com-pared to Pikovskayas medium, has been formulated byNautiyal [39].

After in vitro selection of the most efficient phosphatesolubilizers, experiments should be carried out in order

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to know the effectiveness of the selected PSB strain(s) inassociation with the crop to be inoculated. The responseof plant species or genotypes to inoculation often variesaccording to the bacterial strain used. Differential rhizos-

phere effect of crops in harboring a target PSB strain[15, 41] or even the modulation of the bacterial phos-phate-solubilizing capacity by specific root exudates [20]may account for the observed differences.

Finally, field trials to test the performance of the inoc-ula under real conditions are advisable since the efficien-cy of the inoculation varies with the soil type, P contentof the soil, and other parameters [51]. For example, inthe former Soviet Union a commercial biofertilizer underthe name phosphobacterinwas first prepared by incor-porating Bacillus megaterium var. phosphaticum andwidely used in the Soviet Union, East European coun-tries and India with successful results [58]. However, it

did not show the same efficiency in soils in the UnitedStates [57].

4. CHARACTERIZATION OF PHOSPHATE-SOLUBILIZING BACTERIA

Selected bacteria capable of persistent and high phos-phate solubilization can be identified by phenotypiccharacterization or genotypic characterization.Phenotypic characterization relies mainly on numerousbiochemical tests and was the traditional way to identifybacteria until the application of new molecular tech-

niques for classification and description of microorgan-isms. Several commercial kits make this work easier. Forexample, the BIOLOG system can identify a wide rangeof soil bacteria (BIOLOG, California, USA;http://www.biolog.com), API 20NE system (bioMrieux,France; http://www.biomerieux.com) allows the identifi-cation of species among the pseudomonads group(including Acinetobacter) and the IdentBacillus system(Mikrokit Ibrica S.L., Spain; http://www.bme.es/microkit) is able to differentiate between bacilli species.Nevertheless, to deal with the very complex and scarcelyknown microbial soil populations, molecular techniquesare, without doubt, the most feasible tools.

Currently, the 16S rRNA sequence is by far the pre-ferred phylogenetic marker used in microbial ecology[40]. As molecular chronometers, rRNA sequences havepreserved their evolutionary history [68]. Highly con-served portions carry the information of early evolution-ary events and changes that are more recent occur withinless conserved positions or stretches. The degree ofdivergence of present day rRNA sequences gives an esti-mate of their phylogenetic distances. PCR amplificationof bacterial 16S rRNA sequence is carried out using uni-

versal bacterial primers, such as fD1 (5 -CCGAATTCGTCGACAACAGAGTTTGATCCTG-GCTCAG-3) and rD1 (5-CCCGGGATCCAAGCT-TAAGGAGGTGATCCAGCC-3) [67]. The obtained

16S rDNA amplicons are sequenced, and comparativeanalysis using comprehensive databases of bacterial 16SrRNA gene sequences (http://rdp.cme.msu.edu/html;http://www.ncbi.nlm.nih.gov/Banklt) with appropriatesoftware allows rapid identification of unknown bacteriabased on their rRNA sequence data [34, 35]. However,the power of rRNA sequence based protocols resides at alow phylogenetic or taxonomic level of resolution, whichis valuable for classifying bacteria at the genus level butis insufficient to classify bacteria at the (sub)specieslevel [13, 25, 68]. Other more recent methods overcomethis limitation.

A new, one-dimensional electrophoretic technique in

polyacrylamide gels termed staircase electrophoresis(SCE) has permitted optimum separation of stable low-molecular-weight (LMW) RNA [9]. The LMW RNAprofiles include 5S rRNA and tRNA in prokaryoticmicroorganisms [42, 62, 63, 65] and 5.8S rRNA, 5SrRNA and tRNA in eukaryotic microorganisms [64].These molecules are of great interest for taxonomic pur-poses due to their evolutionary conserved presence in allcells performing the same role (protein synthesis). Thistechnique has already been applied to different groups ofmicroorganisms: the family Rhizobiaceae [62, 65],Frankia [63], Clavibacter[42], and yeasts [64]. A distin-guishing LMW RNA profile was obtained for each bac-terial species assayed. From these works, we can current-

ly affirm that all strains belonging to the same speciesdisplay the same LMW RNA profile (both 5S rRNA andtRNA zones) and all species belonging to the samegenus display the same 5S rRNA zone. Therefore, differ-ent genera can be distinguished by the 5S rRNA zoneand different species can be distinguished by tRNA pro-files (Fig. 1A).

More recently, a new PCR-based procedure, designat-ed as Two Primers (TP)-RAPD fingerprinting, has beendescribed to identify bacteria [50]. This method uses twoprimers, targeting partial sequences of the 16S rRNAgene of Escherichia coli , to obtain species-specificRAPD fingerprints (Fig. 1B). We have demonstrated that

the different subspecies of Clavibacter michiganensisand Pectobacterium (Erwinia) carotovorum can be dis-tinguished by such fingerprints (unpublished data).Therefore, both LMW RNA and TP-RAPD profiles canbe used to identify bacteria at species and subspecieslevels.

Finally, to study the biodiversity of these bacteria,strain-related profiles should be used. Three families ofshort intergenic repeated sequences have been found in

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eubacteria, namely REP (Repetitive ExtragenicPalindromic elements), ERIC (Enterobacterial RepetitiveIntergenic consensus) and BOX elements [66]. The PCRtechnique using specific primers corresponding to theseubiquitous and highly conserved repetitive DNAsequences (REP-, ERIC- and BOX-PCR; collectivelyknown as rep-PCR) has demonstrated to generate strain-specific DNA fingerprints (Fig. 1C). This method ishighly discriminating, allowing the differentiation ofvery closely related bacterial strains within species [10,33, 46, 66]. Hence, it provides a rapid and universal toolfor assessing genomic variation in prokaryotic organ-isms. Studies on microbial ecology have particularlyprofited from these techniques [12, 30]. rep-PCR is auniversal tool for assessing genomic variations inprokaryotic organisms and is postulated to reflect thevariability of the overall genome. No strain-, species-, orgenus-specific primers are involved, allowing the use ofonly a few primers for the analysis of close as well aswidely divergent bacteria. Moreover, no Southern blot-ting or DNA hybridization experiments are required toanalyse the PCR products, and simple agarose gels aresufficient to separate them. Therefore, in studying micro-

bial biodiversity, rep-PCR genomic fingerprinting is theleast experimentally demanding method of choice, espe-cially when complemented with computer-assisted pat-tern analysis.

5. OBTAINING STRAIN-SPECIFIC DNA PROBESBY REP-PCR BASED TECHNIQUES

Good competitive ability and high saprophytic com-petence are the major factors determining the success ofa bacterial strain as an inoculant [55]. Studies to discoverthe competitiveness and persistence of specific microbialpopulations in complex environments, such as the rhi-zosphere, require methods for detecting and estimatingthe relative abundance of specific strains. The use ofDNA probes for detection of soil-borne, specific phylo-genetic groups has yielded promising results. However,laborious procedures are needed to obtain strain-specificprobes [8, 36, 54, 56].

A method has been recently developed by Mathesonet al. [37] to obtain strain-specific DNA probes fordetecting bacteria in the environment based on rep-PCR

Figure 1. SCE LMW RNA profiles (A), TP-RAPD fingerprints (B) and REP-PCR fingerprints (C) of three phosphate-solubilizingrhizobia: lane 1, Sinorhizobium meliloti ATCC 9930T [22]; lane 2,Rhizobium leguminosarum ATCC 10004T [24]; and lane 3,R. etliCFN 42T [Encarna Velzquez, personal communication]. Lane (MW) molecular size markers (ATCC: American Type CultureCollection; CFN: Centro de Fijacin de Nitrgeno, Cuernavaca, Mexico).

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techniques (Fig. 2), which produce, for each bacterialstrain, a unique collection of DNA fragments that differin size and, hence, in sequence. Briefly, the methodemploys amplification of genomic DNA by rep-PCR

(Fig. 2a), followed by cloning of the amplified fragmentsin Escherichia coli competent cells through a plasmidvector (Fig. 2b). After purification (Fig. 2c), the clonedfragments are labeled with digoxigenine (DIG)-dUTP.Finally, these probes are screened against Southern blotsof rep-PCR-amplified DNA fragments from both axenicbacterial cultures and environmental samples of micro-bial communities in order to identify those that arestrain-specific (Fig. 2d). The high sensitivity of theseprobes allows the detection of extremely low numbers oftarget bacteria among a very complex and rich mixtureof nontarget strains. These probes have sensitivity com-parable to those of other PCR-based detection methods.

The method developed by Matheson et al. [37] hasseveral advantages over other approaches for obtaining astrain-specific molecular probe: (i) the probe can bedeveloped and used without any prior knowledge of thehost strain or the probe sequence, (ii) the target sequencecan be amplified from a mixed community without theneed for strain-specific PCR primers, and (iii) a semi-quantitative estimate of the number of target organismspresent in a sample can be made by combining rep-PCRwith a standard most-probable-number (MPN) procedure[48]. Studies on microbial ecology of phosphate-solubi-lizing bacteria may take advantage of the use of such

probes. Due to the fact that many countries have strictregulations concerning the release of genetically engi-neered microorganisms, this methodology excels inapplicability to those that use reporter genes, such as the-glucoronidase gene (gusA) or the thermostable -glu-cosidase gene (celB) [55], as they imply genetic engi-neering of microorganisms.

6. CONCLUSIONS

Several recently developed molecular techniquesallow us, in a rapid and easy way, to identify and detectphosphate-solubilizing bacteria. Studies on biodiversity,distribution and ecology of such bacterial populations insoil and plant rhizospheres may help to design efficientbiofertilizers for each crop and soil type, which can con-tribute to improving farmersprofit by reducing theapplication of chemical fertilizers and, thus, to preserv-ing environmental quality.

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