molecular and biochemical aspects of rhizobacterial ecology with emphasis on biological control

Review

Molecular and biochemical aspects of rhizobacterial ecologywith emphasis on biological control

Veena Kumari* and J.S. SrivastavaInstitute of Agricultural Sciences BHU, Varanasi-221005, India*Author for correspondence: c/o Mr. T.C. Gupta, Diesel Components Pvt. Ltd., 6, Industrial Estate,Jagdish Patti, Jaunpur-222001, India. Tel.: 05452-62902; 05452-62795, Fax: 0542-370251

Received 28 May 1999; accepted 16 June 1999

Keywords: Biochemical, biological control, molecular, rhizobacteria.

Summary

The rhizosphere is the narrow zone of soil surrounding the root that is subject to in¯uence by the root.Rhizobacteria are plant-associated bacteria that are able to colonize and persist on roots. An understanding of theecology of a microorganism is a fundamental requirement for the introduction of a microbial inoculant into theopen environment. This is particularly true for biological control of root pathogens in the rhizosphere, where one isactively seeking to alter the ecological balance so as to favour growth of the host plant and to curtail thedevelopment of pathogens. Some strains of plant growth-promoting rhizobacteria can e�ectively colonize plantroots and protect plants from diseases caused by a variety of root pathogens and growth promotion of plantsthrough direct stimulation of growth hormone. Such bene®cial or plant health-promoting strains are emerging aspromising biocontrol agents. They are suitable as soil inoculants either individually or in combination and may becompatible with current chemical pesticides. Considerable progress has been achieved using molecular genetictechniques to elucidate the important microbial factors or genetic traits involved in the suppression of fungal rootdiseases. Strategies utilizing molecular genetic techniques have been developed to complement the ongoing researchranging from the characterization and genetic improvement of a selected biocontrol agent to the measurement of itspersistence and dispersal. Finally, biocontrol is considered as part of a disease control strategy like integrated pestmanagement which o�ers a successful approach for the deployment of both agro-chemicals and biocontrol agents.

The dramatic worldwide increase in agricultural andindustrial productivity has created severe environmentalproblems. For instance, soil and ground water reservoirshave been polluted with pesticides, xenobiotics andheavy metals. Biological agents such as bacteria withbene®cial properties (as plant growth promotingstrains), could in some cases substitute for pesticides(biological control), or serve to remediate contaminatedenvironments like soil and sediment. Bacteria havealready been introduced into soil to promote plantgrowth (De Freitas & Germida 1992) for pest control(Ross et al. 1998) or for the degradation of a variety ofpolluting compounds (Shao & Behki 1996). Althoughmany bacteria are naturally capable of performingspeci®c functions, the range of possibilities is limited,since introduced bacteria do not always survive andperform well in the soil ecosystem. Bacteria isolatedfrom the environment, which are potentially betteradapted to ecological stresses, can be genetically alteredfor a speci®c environmental purpose (Kusian & Bowien1997). The use of recombinant microorganisms in large-scale ®eld trails is, however, still restricted. This is due tolack of knowledge on the fate of the microorganism and/

or the heterologous DNA and its possible ecological andhealth e�ects as a consequence of the release (Van derBij 1996). One area of particular concern is the transferof introduced genes to indigenous microbes. There isevidence which suggests that environmental transfor-mation, transduction and conjugation take place underfavourable conditions (Wellington & Van Elsas 1992),since soil is a dynamic environment with special char-acteristics for interactions between its bacterial inhab-itants. Many environmental releases of geneticallyengineered bacteria will be into soil e.g., in biologicalcontrol (Krauss & Loper 1995; Nastch et al. 1997), inbioremediation (Bardiya & Chaudhary 1998), in forestry(Reddy & Funk 1997) and in biomining, focussed on theconduciveness of soil to genetic interactions betweenintroduced and indigenous bacteria.

Soil and Rhizosphere

Soil represents a three-phase environment composed ofsolid, liquid and gaseous phases. The solid phase isstatic, as opposed to the liquid and gaseous phases,

World Journal of Microbiology & Biotechnology 15: 535±543, 1999. 535Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.

where conditions are dynamic and commonly ¯uctuate.The solid phase contains inorganic substances such asclay, silt, sand and organic matter which generally aredistributed unevenly and are often complexed in aggre-gates of varying sizes, composition and stability. Ag-gregates important for soil microorganisms are clayorganic matter complexes, due to their negativelycharged surfaces and increased nutrient availability(Smiles 1998). The solid phase is interspersed with thesoil pore network containing liquid and gaseous phasesof varying composition. The volume of pores withdi�erent sizes is governed by soil structure and textureand determines the moisture retention capacity of thesystem, and thereby the availability of water for soilmicroorganisms. Bacteria in soil are located in the oftendiscontinuous soil pores, and are commonly associatedwith soil surfaces. In the absence of transporting agentssuch as (multidirectional) water ¯ow, growing plantroots or burrowing soil animals, bacterial movement insoil over large distances is limited (Trevors et al. 1990).Consequently, bacteria in soil are commonly studied ona large scale which may not provide information onindividual bacterium±pore relationships. Bulk soil canbe regarded as an oligotrophic environment, since it isgenerally poor in readily available organic carbon whichprecludes much bacterial growth and activity, and isestimated to be su�cient for only a few cellular divisionsof soil microorganisms per year (Shields et al. 1973).Plant roots are major sites of input of carbon into soil

(Lynch & Whipps 1990). Both water soluble compoundsand insoluble ones like remnants of root cortex cells, arereleased into the rhizosphere. Root-released organics aremore likely to be decomposed by soil bacteria thancarbon present in the bulk soil due to a lower degree ofrecalcitrance (Dashti et al. 1997). Therefore, the rhizo-sphere can be characterised as a region in soil with ahigh availability of carbon as well as other nutrientssuch as N, P and S compounds. Structural cellularmaterial liberated upon root death provides a morerecalcitrant carbon source. Rhizobacteria often showincreased growth and activity due to the enhancedavailability of organic and inorganic carbon in therhizosphere (Cramer & Richards 1999). Moreover,water ¯ow in soil induced by roots may enhancebacterial movement towards them. Both mechanismsmay promote cell±cell, cell±bacteriophage and cell±DNA contacts, which pictures the rhizosphere as anarea in soil potentially conducive to genetic processesbetween soil bacteria (Sviel et al. 1998). All known genetransfer mechanisms, transformation, transduction andconjugation, are controlled by speci®c conditions thata�ect key bacterial parameters such as cellular physiol-ogy, population density and possibly cellular movementand establishment. These parameters a�ect the fateand survival of parent strains and potential excipients(transformants, transductants and transconjugants).Since donor and excipient survival and activity is crucialfor the impact gene transfer may have on soil popula-tions, data on how bacterial survival is a�ected by soil

factors are useful in the interpretation of results of genetransfer processes. The e�ects of factors such as soil type(texture), pH, moisture, temperature, clay and organicmatter content and predatory protozoa have beenreviewed (Van Elsas & Trevors 1990).It is generally accepted that PGPR (Plant Growth

Promoting Rhizobacteria) must become positioned onor in the root, or in the rhizosphere to promote growth(Natsch et al. 1997, Harris 1997). Rhizobacteria grow-ing in or near infection sites on roots, as well as inchannels in the rhizosphere that provide physical accessto the root, are ideally positioned to limit the establish-ment and spread of pathogens. Several studies havedemonstrated that PGPR suppress populations of rootpathogens (Natsch et al. 1997). Linear regression anal-ysis demonstrated an inverse relationship between thepopulation size of Pseudomonas ¯uorescens and thenumber of lesions, thus indicating that as colonizationincreased, overall control improved (Bull et al. 1991).Another important question is the duration that popu-lations of PGPR must be maintained in order to closethe `window of vulnerability' to infection by pathogens.In general, the smaller the `window of vulnerability', thegreater are the chance of successful biocontrol.The distribution, multiplication and survival of intro-

duced PGPR are profoundly a�ected by biotic andabiotic factors. Howie et al. (1987) studied the e�ect ofsoil matric potential on colonization of wheat roots byseed-applied P. ¯uorescens in the absence of percolatingwater. The optimal temperature for growth of manyPGPR in vitro is above 25 °C but root colonization isgenerally greatest below 20 °C (Loper et al. 1985).Better root colonization at lower temperature probablyre¯ects the fact that microbial activity in the soil declineswith temperature. Further, slower root growth at lowertemperatures may facilitate more e�ective transport ofthe bacteria from the inoculum source to the roots.Although PGPR grow best in vitro at neutral pH orabove, colonization is better at lower pH, possiblybecause of lower competition.

Root colonization

The biological composition of the rhizosphere dramat-ically in¯uences root colonization. It is well understoodthat in the rhizoplane and rhizosphere nutrient avail-ability rather than space is the primary determinant ofmicrobial population size (Pal et al. 1998). Thus intro-duction of PGPR does not result in a change in the totalrhizosphere population, but a shift in the composition ofthe micro¯ora such that introduced bacteria preemptsestablishment of the normal indigenous strains. Thus,root colonization will be greater in sterile or pasteurizedsoils than in raw soil because there is less competitionantibiosis and predation from the indigenous micro-¯ora. In contrast, as microbial activity increases in thesoil, through inputs of nutrients, the level of coloniza-tion by introduced PGPR is reduced (Caseida 1992).

536 V. Kumari and J.S. Srivastava

Pathogens that are targets of PGPR can in¯uencePGPR populations either positively or negatively(Edwards et al. 1999).Root colonization is a multistage process: undoubt-

edly many bacterial traits and genes are involved. Theimportance of each trait may di�er amongst PGPR.Adhesion of PGPR to roots may be either non-speci®cresulting from electrostatic forces (James et al. 1985), orinvolve speci®c recognition between the surfacesthrough glycoprotein termed as agglutinin (Andersonet al. 1988). Buell & Anderson (1992) characterized alocus, aggA, from Pseudomonas that encodes a predicted50.5 KDa protein required for agglutinability and ad-herence. Several di�erent exopolysaccharides are in-volved in the attachment of rhizobacteria to plant cellsand in the nodulation of legumes by Rhizobium (Can-gelosi et al. 1987). One approach to increasing rootcolonization by PGPR is to increase the dose of thebacteria applied to the seed (Bull et al. 1991). However,increasing colonization by increasing the initial dose ofbacteria on the seed has limitations (Osburn et al. 1989).Another approach to increase colonization and biocon-trol is the application of mixtures of strains. PGPRresearch has focussed primarily on the use of singlestrains. However, Weller & Cook (1983) demonstratedthat P. ¯uorescens 2±79 used in combination withP. ¯uorescens 13±79 was superior to either strain alonein about 50% of the trials. Recombinant DNA tech-nology has provided the most exciting and potentiallysuccessful means to improve root colonization andbiological control by PGPR (Natsch et al. 1997). Im-provements can also be achieved through the transfer ofbiocontrol traits into other strains (Saxena et al. 1998).

Role of marker genes and DNA probes in detectionof bacteria in the rhizosphere

Microorganisms are introduced into the soil and rhizo-sphere to improve plant growth through nitrogen®xation, biological control of disease, formation ofmycorrhizae and direct growth stimulation need tomonitor introduced organisms speci®cally, to study theirfate in the environment and thereby to help explain thesuccess or failure of the inoculation (Edwards et al.1999). It also needs to monitor the fate of geneticallymanipulated organisms after they are released into theenvironment. Virtually all genetically manipulated or-ganisms so far released into the environment have beenmonitored. Antibiotic resistance is useful, especiallywhere the genetic modi®cation itself has not provided aselectable marker (Saxena et al. 1998). In other cases,the use of a spontaneous antibiotic-resistant derivativehas made the selective isolation of the genetically-manipulated organism from the environmental samplemore straightforward (Natsch 1999). There have beennumerous studies of long term survival of rhizobacteriain soil, but there have been few reports of the long termstability of antibiotic resistance markers in biological

control agents (Lindo & Panopoulos 1988; Van der Bijet al. 1997; Nebevoncaron et al. 1998).The use of marker genes involves the addition of new

DNA to an organism so that it can be uniquelyidenti®ed and distinguished from other microorganismsin the environment into which it is introduced (McDer-mott et al. 1997). Scanferlato et al. (1990) monitored agenetically manipulated strain of rhizobacteria by MPN(Most Probable Number) analysis. Lactose utilization(lac ZY from E. coli) has been introduced into Pseudo-monas ¯uorescens as a plasmid based on a Tn7-mediatedchromosomal marker (Drahos et al. 1986). Resistanceto heavy metals (Mercury) as marker is encoded by themer operon of rhizobacterium (Herrero et al. 1990) formontioring. Whole or part of the lux operon has beenused to monitor not only the survival and distribution ofintroduced bacteria (Kozdraj 1997) but also the activityof the organism (Meikle et al. 1992). Resistance to theherbicide bialaphos is encoded by the bar gene (Herreroet al. 1990). Ramos et al. (1991) used the ptt markertogether with plasmid-coded p-ethylbenzoate degrada-tion to speci®cally select a Pseudomonas strain.Tranoposons which code for antibiotic resistance havebeen used frequently to monitor introduced organismsin soil and rhizosphere (Bentjen et al. 1989; Spreg &Gentschev 1998).Bacteria can be detected in the rhizosphere by the use

of speci®c probes to DNA or RNA sequences (Napo-leao et al. 1998). A DNA probe is normally a shortDNA sequence that matches and will bind uniquely toDNA of a particular organism or group of organisms,depending on the level of speci®city desired. Syntheticnucleotide sequences can be inserted into the genomeand used with speci®c DNA probes to detect theorganism (Amici et al. 1991). Probes can also bespeci®cally targeted to RNA (Amann et al. 1990).Ampli®cation of the target DNA sequence has beenachieved by use of the polymerase chain reaction (PCR).The use of ampli®cation by PCR has been reviewed bySte�an & Atlas (1991) and Cullen et al. (1998). The useof ampli®cation by PCR the possible deleterious e�ectsof genetic manipulation on soil bacteria include: (a) the`metabolic loss' of carrying added genetic material andthe expression of foreign genes, (b) loss of competitiveability and (c) decreased ®tness. A deleterious e�ectwhich is possibly due to metabolic load, is the decreasedbiological control activity of an antibiotic-overproduc-ing mutant of rhizobacterium (Shim et al. 1987). Takingthe lac ZY-marking of pseudomonads, Drahos et al.(1992) reported no di�erence in long-term survival in the®eld when comparing the engineered strain to the parentstrain Pseudomonas (Srivastava 1990). The engineeredorganisms can usually be tracked readily, because thealteration can be detected by using nucleic acid probes.The ability to track both genetically-engineered andnon-modi®ed bacteria is increasingly necessary forboth ecological studies of their fate after release, forthe accumulation of data that can be used in riskassessment.

Biological control in rhizobacteria 537

Role of genetic strategies in environmental signals

Genetically engineered rhizobacteria destined for delib-erate release for environmental and agricultural appli-cations, should express their recombinant genes underthe control of signals present in the location where thebacteria are expected to operate (De Lorenzo 1992). Thetype of signals vary, depending on the speci®c situationand in some cases they might not be completely de®nedphysically or chemically. A Pseudomonas strain designedto biodegrade polychlorobiphenyls (PCBs) in the rhizo-sphere of certain plants (Walton & Anderson 1992)might be desired to activate transcription of the cognatecatabolic pathway only in the vicinity of root exudates,the composition of which might not be well known.Similarly conditional suicide genetic circuits (Molinet al. 1987) might be desired, which activate lethal genesto avoid proliferation of the genetically engineeredmicroorganisms during a certain season of the year(i.e., in a temperature-dependent fashion). Althoughconstruction of e�cacious expression systems for strainsperforming in such di�erent conditions can be addressedon a case-by-case basis, the potential demand of speci®c-purpose recombinant microorganisms in the future hasencouraged the development of new broad-host-rangeexpression devices speci®cally tailored for bacteria inuncontained applications.Transposon-vectors (Spreng & Gentschev 1998) are

also utilized as the basic unit to assemble additionalgenetic traits. Transposons are among the most usefultools available to the microbial geneticist. The spectrumof di�erent types of transposon, their mechanisms oftransposition and their applications in genetic engineer-ing, have been reviewed by Berg et al. (1989). Tn5 is acomposite transposon, i.e., its mobility is determined bytwo insertion sequences (IS50) ¯anking the DNA regiondetermining the selectable phenotype. Tn5 can betransferred among replicons as a consequence of theaction of the transposase, Tnp, encoded by IS50R, oncognate short target sequences located at the end of thetransposon. While the Tnp gene is a component of thenaturally occuring Tn5, its product still works when thegene is arti®cially placed outside of the mobile unit,though preferably placed in cis to the cognate terminalsequence (Berg et al. 1989). The construction of recom-binant transposons in which only those elements essen-tial for transposition (i.e., IS terminal sequence andtransposase gene) have been retained and are arrangedsuch that the transposase gene is adjacent to but outsidethe mobile DNA segment. Since the elimination of non-essential sequences leads to a major reduction oftransposon size, the resulting recombinant mini-Tn5transposons are much more convenient to handle thannatural transposons. Two properties of mini-Tn5 deriv-atives are released. First, once inserted into a targetsequence, mini transposons are stably inherited and donot provoke DNA rearrangements or other forms ofgenetic instability. This is due to the lose of the cognatetransposase gene and the greater parts of the IS

(insertion sequence) elements present in the wild typetransposon. A second property is that due to the loss ofthe transposase gene during transfer to a new replicon,the host cell does not become immune to further roundsof transposition. This permits the organism to bereinserted with the same system provided that thesubsequent transposons contain a distinct selectionmarker (Herrero et al. 1990). The simple organizationof mini-transposons has made them a sort of `Swissarmy knife' to construct GEMs destined for environ-mental applications. The possibility now exists not justto release into the environment predictably modi®edlaboratory strains, but to improve the phenotype ofnaturally occurring bacteria by inserting into theirchromosome one or more hybrid transposons througha very simple mating protocol (De Lorenzo et al. 1990;Srivastava 1995).Another foreseeable area of vector development is the

utilization of starvation promoters for heterologousexpression purposes in the ®eld. Since growth of soilbacteria occurs under nutrient conditions unable tosupport exponential growth, nutrient starvation isconsidered a universal signal, potentially useful toexpress heterologous genes in the environment (Murphyet al. 1998). Promoters responsive to carbon, nitrogen,iron and phosphate starvation have been characterizedin many gram-negative bacteria and they are in principleexcellent assets as building blocks for expression systems(Matin 1991). An alternative sigma factor seems to bedirectly or indirectly involved in expression during lategrowth stages and stationary phase. Random insertionin front of growth phase-dependent promoters give riseto exconjugant colonies with distinct phenotypes (deLorenzo et al. 1993). Further developments of thesystem for heterologous expression purposes includethe construction of a plasmid capable of delivering,through homologous recombination, a promoterlessgene (or genes) in front of the starvation promoter,and its combination with the t7 polymerase gene.

Current concepts of biological control

Plant growth-promoting rhizobacteria (PGPR) are usedas model organisms to study not only the mechanisms ofdisease suppression but also the ecological impact ofintroduced plant-bene®cial bacteria. Biological controlof root diseases with wild type or genetically modi®edmicroorganisms requires the deliberate release of thesemicroorganisms in large number into soil ecosystems.Although plant growth-promoting rhizobacteria havenot been observed to cause damaging e�ects on theenvironment it requires approaches to determine theecological competence (e.g., root colonization, survivalin the rhizosphere and in neighbouring soil) of releasedmicroorganisms, their dissemination (e.g., to groundwater) and their impact on resident microbial popula-tions (Araujo et al. 1996; Bent & Chanway 1999).Environmental monitoring of PGPR strains can be


done by using (1) speci®c polyclonal antibodies directedagainst the cell wall of PGPR strains that have beenlabeled with ¯uorescein-isothiocyanate (2) the typicalpro®le of secondary metabolites (3) Southern hybrid-ization of genomic DNA with phl gene probes (Vincentet al. 1991; Spreng & Gintschev 1998).Metabolities produced and excreted by P. ¯uorescens

are assumed to be important and biotic factors in thebiological control of root diseases (O'Sullivan & O'Gara1992; Kloepper et al. 1980). These products can bebroadly classi®ed into two groups: Siderophores andsecondary metabolites. The siderophores pyoverdine(pseudobactin), salicylate and pyochelin are all producedby ¯uorescent pseudomonads when these bacteria aregrown under iron-limiting conditions (Krauss & Loper1995). These three siderophores might give these bacte-ria a competitive advantage over other rhizospheremicroorganisms, especially pathogens, in iron acquisi-tion (Roleto et al. 1999). When growth slows down andcells enter the stationary phase, Pseudomonas ¯uorescensproduces a battery of secondary metabolites e.g. hydro-gen cyanide (HCN). The other metabolites are indole-acetic acid (IAA), 2,4-diacetylphloroglucinol (DAPG),pyoluterin (plt) and pyrrolnitrin. The biological prop-erties of these secondary metabolites are diverse. Cya-nide is toxic in general because it forms stable complexeswith several divalent metal ions. In particular, cyto-chrome oxidase of many of organisms, is stronglyinhibited by cyanide (Way et al. 1988). Pseudomonas¯uorescens and a variety of other microorganisms,however are relatively insensitive to cyanide, they havean alternate cyanide-resistant cytochrome oxidase. Theconcentrations of HCN (»100 lM) that are producedin vitro by Pseudomonas ¯uorescens cause growth stimu-lation or promotion of the producer strain and other¯uorescent pseudomonads but inhibit growth of somefungus (Ahl et al. 1986; Munimbazi & Bullerman 1998;Edwards et al. 1998). IAA, a plant growth hormonewhose synthesis is initiated by L-tryptophan side chainoxidase is induced in the stationery phase in Pseudo-monas ¯uorescens. The quantities of IAA in Pseudo-monas ¯uorescens are lower than those observed forsome other rhizosphere microorganisms e.g., Azospir-illum spp. (Oberhansli et al. 1991). N(b-Ketocaproyl),L-homoserine lactone (KHL) produced by Pseudomonas¯uorescens is better known as the autoinducer ofbioluminescence in marine bacterium (Vibro ®scheri)which when it reaches a certain cell density in batchculture, the autoinducer accumulates in the medium.Above )1 lg/ml concentration, KHL trigers transcrip-tion of the lux genes encoding the enzymes involved inbioluminescence (Meighen 1991; Bainton et al. 1992).Besides autoinducer, environmental signals (e.g. low O2

and Fe levels) contribute to the expression of biolumi-nescence (Williams et al. 1992) and are implicated as animportant signal of secondary metabolism and virulencefactors in rhizobacterial strains and other bacterialspecies (Bainton et al. 1992). KHL is structurally similarto an extracellular autoregulator of streptomyces spe-

cies, termed A-factor, which is an essential signal forantibiotic synthesis and cellular di�erentiation (Beppu1992). A fascinating picture is emerging: many micro-organisms, when they reach high cell densities, producesmall quantities of chemical signals which trigger veryspeci®cally a range of cellular functions: di�erentiationantibiosis, symbiosis, or virulence (Beppu 1992).Iron is an essential nutrient for most microorganisms.

However, it is probably unique in its near insolubility inaerobic environments at pH 7 (Neilands 1984). Thisimposes a severe limitation on its availability. Toovercome this, most microorganisms secrete low molec-ular weight, high a�nity iron-chelating compoundstermed siderophore. Pseudomonas strains usually pro-duce a siderophore consisting of a ¯uorescent quinolinegroup covalently linked to a peptide in which two of theamino acid side chains are modi®ed for iron chelation.There is typically a strain-dependent variation in thestructure of the siderophore produced. They are gener-ally referred to as pseudobactins or pyoverdine-typesiderophores. The component of the standard microbialiron transport system is a receptor that speci®callyrecognizes the ferric complex of the siderophore foruptake of the metal. In gram-negative bacteria, suchreceptor proteins are found in the outer memberane(Neilands 1982). Research in biological control hasfocussed on the potential of using or modifying bacterialiron transport systems to enhance the abilities of plantgrowth-promoting rhizobacterial strains (Morris et al.1992). The expression of iron-regulated genes, such assiderophore biosynthesis and siderophore receptorgenes, is modulated by the level of available environ-mental factors. In Pseudomonas M114, iron-regulatedgene expression is modulated by both positive andnegative regulatory elements (O'Sullivan & O'Gara1990) Under conditions of iron limitation, this gene(pbrA) is required for iron-regulated gene expression.Transcription of iron-regulated genes is blocked underconditions of iron su�ciency. In E. coli a consensusDNA sequence to the coding sequences of iron-regu-lated genes has been shown to be the site at which therepressor binds to inhibit transcription (deLorenzo et al.1987). Because similar `Iron box' sequences are found 5¢to pbrA from Pseudomonas M114 it may indicate thatpbrA, in turn, is regulated by a fur-like protein inPseudomonas M114 which contains a fur-like gene andsuch a gene has been isolated and sequenced from otherrhizobacterial strain (Pseudomonas aeruginosa) (Princeet al. 1993). This may lead to the engineering of diverserhizosphere strains with improved iron assimilationability and inoculation with improved biocontrol andcompetitive properties (Saxena et al. 1998; Yurgel et al.1998).Another mechanism by which a biocontrol agent acts

to protect plants from pathogens depends on theproduction of antifungal metabolities (O'Sullivan &O'Gara 1992; Munimbazi & Bullarman 1998). Theproduction of the antimicrobial compound, 2,4-diace-tylphloro-glucinol (DAPG) by Pseudomonas ¯uorescens


is also an important factor in the control of black rootrot on tobacco (Keel et al. 1990). A desirable goal is toconstruct improved biocontrol agents by means ofincorporation of additional factors that antagonize thegrowth of a target pathogen into a single strain. Theconversion of MAPG to DAPG, the chromosomalregion from F113 encoding was introduced into a seriesof rhizosphere pseudomonad isolates and only Pseudo-monas M114 was capable of DAPG production (Fentonet al. 1992). In the case of genetic instability, a positiveselective pressure may have to be maintained through-out the life cycle of the GEM (Genetically EngineeredMicroorganism) to maintain the engineered trait. Theplasmid vector (PGD11) contains a copy of the Lac-tococcus lactis thymidylate synthase (thyA) gene and thethyA gene product is a key enzyme in de novo DNAsynthesis and essential for growth. The combination of arhizobacterium thy host and PGDT11 ensures absoluteplasmid stability under laboratory, green house and ®eldconditions. Therefore, PGDT11 and similar plasmidsprovide a suitable vector system to introduce genes ofinterest into inoculant strains and ensure stability of theGEM throughout the life cycle of the strain (Ross et al.1990; Kozdroj 1996).

Current concepts in genetic typing of microorganisms

In areas like epidemiology and ecology, as well asbiological safety, it is important to identify species andstrains of organisms accurately. Typically identi®cationand typing of bacterial isolates is done by characterizingphenotypic properties. Utilization of di�erent sub-strates, the presence or absence of certain enzymes,immunological cross-reactivity, phage or antibiotic sen-sitivity and isoenzyme comparisons are commonly usedto classify strains. Recently, methods based on genomeanalysis of organisms are gaining increasing attention.These methods are not in¯uenced by varying growthconditions or culture age which may alter the synthesisof enzymes, metabolities or surface components or theiraccessibility for detecting purposes.In bacteria, VNTRs (Variable Number of Tandem

Repeats) are not found but sequence variabilities dooccur due to a single base change. Thus, RFLP(Restriction Fragment Length Polymorphism) analysismay be useful as a genetic typing for bacterial strains. Auseful class of bacterial RFLP ®ngerprinting detectssequence variations within the operons coding forribosomal RNAs which contain highly conserved aswell as variable regions. The conserved regions are usedto detect DNA restriction fragments containing RNAgenes, whereas the variable regions provide the basis forthe RFLPs (Grimont & Grimont 1986).A more general approach for genetic typing of strains

of any prokaryotic and eukaryotic species is the RAPD(Random Ampli®ed Polymorphic DNA) analysis. ShortPCR primers of arbitrarily chosen sequence are used toamplify a region of the genome which, after separation

of the resulting DNA fragments, produces a bandedpattern useful as a strain-(or individual-) speci®c `®n-gerprint' of the genome (Bassam et al. 1992). A similarapproach using tRNA consensus sequences as PCR-primers generates species-speci®c fragment patterns forbacterial (Cullen et al. 1998), which can be used toassign members of the same genus to a species, afterwhich strains can be further analysed by applying highresolution RAPD ®ngerprinting (Seal et al. 1992).

Conclusion

In general competition for nutrients supplied by rootsand seeds and occupation of sites favoured for coloni-zation probably are responsible for a small or moderatedegree of disease suppression by most PGPR and are ofprimary importance in some strains. Production ofmetabolites such as antibiotics, siderophores and hy-drogen cyanide is the primary mechanism of biocon-trol. Production of secondary metabolites such asphenazine-1-carboxylic acid (PCA), 2,4-diacetyl phlor-oglucinol (phl), pyoleutorin (plt), pyrrolnitrin, oomycinand hydrogen cyanide (HCN) are also the characteristicfeatures of biocontrol agents. The basic strategy that iswidely employed for determining the role of a speci®cgene or trait in a biocontrol process by PGPR involves:(1) development of an assay to demonstrate biocontrolactivity, (2) selection of wild-type strains with biocon-trol, (3) mutagenesis of strains, (4) screening of mutantsfor loss of the desired trait, (5) preparation of a genomiclibrary from wild-type DNA, (6) complementation ofmutants to restore the desired trait. Phenazine-1-car-boxylic acid (PCA) and 2,4-diacetyl phloroglucinol(phl) are the most extensively studied metabolites.There has been considerable research and progress in

the last 20 years in the understanding of the process ofroot colonization and mechanisms of growth-promotionby PGPR. Although, PGPR technology is slowly beingintroduced into agriculture, many impediments still existto the widespread commercialization and use of PGPR.Research must intensify because this technology will bechallenged in the near future to ®ll a void as the use ofchemical pesticides becomes more restricted in agricul-ture. Emphasis is needed on identifying soil edaphicfactors that a�ect biocontrol activity and root coloni-zation, as well as those traits that contribute torhizosphere competence. Also critical is the developmentof uniform and scienti®cally based guidelines for therelease of genetically engineered PGPR (Plant growth-promoting-Rhizobacteria) in order to facilitate moreroutine screening in the ®eld.

Acknowledgement

The author thanks Prof. D.K. Arora (Department ofBotany, Banaras Hindu University, Varanasi) for help-ful suggestions and discussion in preparing the manu-


script. The author also thanks to CSIR, New Delhi for®nancial support during the research period.

References

Ahl, P., Voisard, C. & Defago, G. 1986 Iron bound-siderosphores,

cyanide, and antibiotic involved in suppression of Thielaviopsis

basicola by a Pseudomonas ¯uorescens strain. Journal of Phyto-

pathology 116, 121±234.

Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereus, R.

& Sttahl, D.A. 1990 Combination of 16S rRNA-targeted oligo-

nucleotide probes with ¯ow cytometry for analyzing mixed

microbial populations. Applied & Environmental Microbiology

56, 1919±1925.

Amici, A., Bazzicalupo, M., Gallori, E. & Rollo, F. 1991 Monitoring a

genetically engineered bacterium in a freshwater environment by

rapid enzymatic ampli®cation of a synthetic DNA `number-plate?'

Applied Microbiology & Biotechnology 36, 222±227.

Anderson, A.J., Habibadegah-Tari, P. & Tepper, C.S. 1988 Molecular

studies on the role of a root surface agglutinin in adherence and

colonization by Pseudomonas putida. Applied & Environmental

Microbiology 54, 375±380.

Araujo, M.A.V., Mendonca, Hagler, L.C., Hagler, A.Na. & Van

Elasas, J.D. 1996 Selection of rhizosphere competent Pseudomonas

strains as biological agents in tropical soil. World Journal of

Microbiology & Biotechnology 12, 589±593.

Bainton, N.J., Stead, P., Chhabra, S.R., Bycroft, B.W., Salmond,

G.P.C., Stewart, G.S.A.B. &Williams, P. 1992N-(3-oxohexanoyl)-

L-homoserine lactone regulates carbapenem antibiotic production

in Erwinia carotovora. Biochemical Journal 288, 997±1004.

Bardiya, N. & Chaudhary, K. 1998 Methane recovery from industrial

wheat bran residue. Indian Journal of Microbiology 38, 25±28.

Bassam, B.J., Caetano-Anolles, G. & Greasho�, P.M. 1992 DNA

ampli®cation ®ngerprinting of bacteria. Applied Microbiology &

Biotechnology 38, 70±76.

Bent, E. & Chanway, C.P. 1999 The growth-promoting e�ects of a

bacterial endophyte on lodgepole pine are partially inhibited by the

presence of other rhizobacteria. Canadian Journal of Microbiology

44, 980±988.

Bentjen, S.A., Fredrickson, J.K., Van Voris, P. & Li, S.W. 1989 Intact

soil-core microcosms for evaluating the fate and ecological impact

of the release of genetically engineered organisms. Applied &

Environmental Microbiology 55, 198±202.

Beppu, T. 1992 Secondary metobolites as chemical signals for cellular

di�erentiation. Gene 115, 159±165.

Berg, C.M., Berg, D.E. & Groisman, E.A. 1989 Transposable elements

and the genetic engineering of bacteria, In:Mobile DNA. American

Society for Microbiology. ed. Berg, D.E. & Howe, M.M. Wash-

ington, D.C. 879±926. ISBN 1-55581005-5.

Bull, C.T., Weller D.M. & Thomashow L.S. 1991 Relationship

between root colonization and suppression of Geaumannomyces

graminis var tritici by Pseudomonas ¯uorescens strain 2-79.

Phytopathology 81, 954±959.

Buell, C.R. & Anderson, A.J. 1992 genetic analysis of the aggA locus

involved in agglutination and adherence of pseudomonas putida,

a bene®cial ¯uorescent pseudomond. Molecular Plant Microbe

Interactions 5, 2462±2470.

Cangelosi, G.A., Hung, L., Puvanesara-jah, V., Stacey, G., Ozga,

D.A., Leigh, J.A. & Nestor, E.W. 1987 Common loci for

Agrobacterium tumifaciens and Rhizobium meliloti exopolysaccha-

ride synthesis and their roles in plant interactions. Journal of

Bacteriology 167, 2086±2091.

Casida, L.E. 1992 Competitive ability and survival in soil of

Psuedomonas Strain 679-2, a dominant, nonobligate bacterial

predator or bacteria. Applied & Environmental Microbiology 58,

32±37.

Cullen, D.W., Nicholson, P.S., Mendum, T.A. & Hirsch, P.R. 1998

Monitoring genetically modi®ed rhizobia in ®eld soils using the

polymerase chain rection. Journal of Applied Microbiology 84,

1025±1034.

Cramer, M.D. & Richards, M.B. 1999 The e�ect of rhizosphere

dissolved inorganic carbon on gas exchange characteristics and

growth rates of tomato seedlings. Journal of Experimental Botany

50, 79±87.

Dashti, N., Zhang, F., Hynes, R. & Smith, D.L. 1997 Application of

plant growth-promoting rhizobacteria to soybean (Glycine mox [L]

Merr) increases protein and dry matter yield under short-season

conditions. Plant & Soil 188, 33±41.

De Freitas, J.R. & Germida, J.J. 1992 Growth promotion of winter

wheat by ¯uorescent pseudomonads under ®eld conditions. Soil

Biology & Biochemistry 24, 1137±1146.

De Lorenzo V.S., Herrero, W.M. & Neilands, J.B. 1987 Operator

sequence of the Agrobacterium operon of plasmid col. v-K30

binding the ferric uptake regulation (fur) repressor. Journal of


De Lorenzo, V., Herrero, M., Jacubzik U. & Timmis, K.N. 1990

Mini-Tn5 transposon derivatives for insertion mutagenesis, pro-

moter probing and chromosomal insertion of cloned DNA in

Gram-negative eubacteria. Journal of Bacteriology 172, 6568±

6572.

De Lorenzo, V. 1992 Genetic engineering strategies for environmental

applications. Current Opinion in Biotechnology 3, 227±231.

De Lorenzo, Cases, V.I., Herrero, M. & Timmis, K.N. 1993 Early and

late response of TOL promoters to pathway inducers: Identi®ca-

tion of postexponential promoters in Pseudomonas putida with

laszZ-tet bicistronic reporters. Journal of Bacteriology 175, 6902±

6907.

Drahos, D.J., Barry, G.F., Hemming, B.C., Brandt, E.J., Kline, E.L. &

Kluepfel D.A. 1992 Spread and survival of genetically marked

bacteria in soil In: Release of genetically-engineered and other

microorganisms. ed. Fry, J.C. & Day, M.J. pp. 147±159. UK.

Cambridge University Press. ISBN 0-521-41756-2.

Drahos, D.J., Hemming, B.C. & McPherson, S. 1986 Tracking

recombinant organisms in the environment. b-glactosidase as a

selectable nonantibiotic marker for ¯uorescent pseudomonads.

Biotechnology 4, 439±444.

Edwards, S.G., Young, J.P.W. & Fitter, A.H. 1998 Interactions

between Pseudomonas ¯uorescens biocontrol agents and Glomus

mosseae, an arbuscular mycorrhizal fungus within the rhizosphere.

FEMS Microbiology Letters 166, 297±303.

Fenton, A.M., Stephens, P.M., Crowley, J., O'Callaghan, M. &

O'Gara, F. 1992 Exploitation of gene (5) involved in 2,4-

diacetylphloro-glucinol biosynthesis to confer a new biocontrol

capability to a Pseudomonas strain. Applied & Environmental


Glick, A. 1995 The enhancement of plant growth by free living

bacteria. Canadian Journal of Microbiology 41, 199±117.

Grimnont, F. & Grimont, P.A.D. 1986 Ribosomal ribonucleic acid

gene restriction patterns as potential taxonomic tools. Annolas de l'

Institut Pasteur/Microbiology. (Paris) 137B, 165±175.

Harris, -A.R. 1997 Identi®cation of bacteria selected for biocontrol of

damping o� disease of plant growth promotion. Australiasian

Plant Pathology 26, 192±194.

Herrero, M., de Lorenzo, V. & Timmis, K.N. 1990 Transposon victors

containing non-antibiotic selection markers for cloning and stable

chromosomal insertion of foreign DNA in gram-negative bacteria.

Journal of Bacteriology 172, 6557±6576.

Howie, W.J., Cook, R.J. & Weller, D.M. 1987 E�ects of soil matric

potential and cell motility on heat root colonization by ¯uorescent

pseudomonads suppressive to take-all. Phytopathology 77, 286±

292.

James, D.W.Jr., Suslow, T.V. & Steiback, K.E. 1985 Relation

between rapid, ®rm adhesion and longterm colonization of

roots by bacteria. Applied & Environmental Microbiology 50,

393±397.

Keel, C., Wirthner, P., Oberhansli, T., Voisard, C., Burger, U., Haas,

D. & Defago, G. 1990 Pseudomonads as antagonists of plant

pathogens in the rhizosphere: role of the antibiotic 2,4-diacetyl-


phloroglucinol in the suppression of black root rot of tobacco.

Symbiosis 9, 327±341.

Kloepper, J.W., Leong, J., Tientze, M. & Schroth, M.N. 1980

Enhanced plant growth by siderophores produced by plant growth

promoting rhizobacteria. Nature 286, 885±886.

Kozdroj, J. 1996 Survival of lux-marked bacteria introduced into soil

and the rhizosphere of bean (Phaseolus vulgaris L.). World Journal

of Microbiology & Biotechnology 12, 261±266.

Krauss, J. & Loper J.E. 1995 Chracterization of a genome region

required for production of the antibiotic pyoluteorin by the

biological control agent Pseudomonas ¯uorescens pf-5. Applied &


Kusion, B. & Bowien, B. 1997 Organization and regulation of Cbbco2assimilation genes in autotrophic bacteria. FEMS Microbiology

Review 21, 135±156.

Lindow, S.E. & Panopoulos, J.J. 1988 Field tests of recombinant ice

Pseudomonas sysingae for biological frost control in potato. In:

Release of Genetically-engineered Microorganisms. ed. Sussman,

M., Collins, C., Skinner, F. & Stewart-Tull, D. pp. 121±138. San

Diego: Academic Press. ISBN 0-12-677522-2.

Loper, J.E., Haack, C. & Schroth, M.N. 1985 Population dynamics of

soil pseudomonads in the rhizosphere of potato. Applied and


Lynch, J.M. & Whipps, J.M. 1990 Substrate ¯ow in the rhizosphere.

Plant and Soil 129, 1±10.

Matin, A. 1991 The molecular basis of carbon starvation-induced

general resistant in Escherichia coli. Molecular Microbiology 5,

3±16.

Maurhofer, M., Keel, C., Schnider, U., Voisard, C., Hass, D. &

Defago G. 1992 In¯uence of enhanced antibiotic production in

Pseudomonas ¯uorescens strain CHAO on its disease suppresive

capacity. Phytopathology 82, 190±195.

McDermott, P.J., Winterman, E.A., Gowland, P. & Gowland, P.C.

1997 Enhanced survival of plasmid containing Escherichia coli in

aquatic microcosmos. World Journal of Microbiology & Biotech-

nology 13, 159±162.

Meighen, D.A. 1991 Molecular biology of bacterial bioluminescence.

Microbiological Review 55, 123±142.

Meikle, A., Kilhan, K., Prosser, J.I. & Clover, L.A. 1992 Lumino-

metric measurement of population activity of genetically modi®ed

Pseudomonas ¯uorescens in the soil, FEMS Microbiology Letters

66, 217±220.

Molin, S., Klema, P., Poulsen, L.K., Biehl, H., Gerdes, K., &

Anderson, P. 1987 Conditional suicide system for containment of

bacteria and plasmids. Biotechnology 5, 1315±1318.

Morris, J., O'Sullivan, D.J., Koster, M., Leong, J., Weisbeek, P. &

O'Gara F. 1992 Characterization of ¯urorescent ferric sideroph-

ore-mediated iron uptake in Pseudomonas sp. Strain M114:

evidence for the existence of an additional ferric siderophore

receptor. Applied & Environmental Microbiology 58, 630±635.

Munimbazi, C. & Bullerman, L.B. 1998 Isolation and partial charac-

terization of antifungal metabolites of Bacillus pumilus. Journal of

Applied Microbiology 84(6).

Murphy, D.V., Fillery, I.R.P. & Sparling, G.P. 1998 Seasonal

¯uctuations in gross N mineralization ammonium consumption

and microbial biomass in a western Austrialian soil under di�erent

land uses. Australian Journal of Agricultural Research 49, 523±536.

Napoleao, R., Romeiro, R.S., Berian, L.O.S. & Barbosa, J.G. 1998 A

bioassay for rapid screening of bacteria antagonistic to Agrobac-

terium tumefaciens. Fitopatologia Brasileira 23, 27±29.

Natsch, A., Keel C., Hebecker, N., Laasik, E. & Defago, G. 1997

In¯uence of biocontrol strain Pseudomonas ¯uorescens GHAO and

its antiobiotic overproducing derivative on the diversity of resident

root colonizing psudomonads. FEMS Microbiology Ecology 23,

341±352.

Natsch, A., Keel, C., Hebecker, N., Laasik, E. & Defago, G. 1999

Impact of Pseudomonas ¯uorescens strain CHAO and a derivative

with improved biocontrol activity on the culturable resistant

community on cucumber roots. FEMS Microbiology Ecology

27(4).

Nebevoncaron, G., Stephens, P. & Badley, R.A. 1998 Assessment of

bacterial viability status by ¯ow cytometry and single cell sorting.

Journal of Applied Microbiology 84, 988±998.

Neilands, J.B. 1982 Siderophores of bacteria and fungi. Microbiolog-

ical Science 1, 9±14.

Neilands, J.B. 1984 Microbial envelope proteins related to iron. Annual

Review of Microbiology 36, 285±309.

O'Sullivan, D.J. & O'Gara, F. 1990 Iron regulation of ferric iron

uptake in ¯uorescent pseudomonas: Cloning of a regulatory gene.

Molecular Plant Microbe Interactions 3, 86±93.

O'Sullivan, D.J. & O'Gara, F. 1992 Traits of ¯uorescent Pseudomonas

Spp. involved in suppression of plant root pathogens. Microbio-

logical Reviews 56, 662±676.

Oberhansli, T., Defago, G. & Haas, D. 1991 Indole-3-acetic acid (IAA)

synthesis in the biocontrol strain CHAO of pseudomonas ¯uo-

rescens: role of tryptophan side chain oxidase. Journal of General


Osburn, R.M., Schroth, M.N., Hancock, J.G. & Hendson, M. 1989

Dynamics of sugar beet seed colonization by Pythium ultimum and

pseudomonas species: e�ects on seed root and damping o�.

Phytopathology 79, 709±716.

Pal, S., Manna, A. & Paul, A.K. 1998 Nutritional and cultural

conditions for production of poly-3-hydroxybutyric acid by

Azotobacter chrococcum. Folia Microbiologica 43, 177±181.

Prince, R.W., Cox, C.D. & Vasil, M.L. 1993 Coordinate regulation of

siderophore & exotoxin A production: Molecular cloning and

sequencing of the pseudomonas aeruginosa fur gene. Journal of


Ramos, J.L., Duque, E. & Ramos-Gonzalez, M.I. 1991 Survival in

soils of an herbicide-resistant pseudomonas putida strain bearing a

recombinant TOL plasmid. Applied & Environmental Microbiology

57, 260±266.

Reddy, M.S. & Funk, L.M. 1997 Microbial inoculants for sustainable

forests. Journal of Sustainable Forestry 5, 293±306.

Roleto, E.A., Borneman, J. & Triplett, E.W. 1999 E�ects of bacterial

antibiotic production on rhizosphere microbial communities from

a culture independent perspective. Applied & Environmental


Ross, P., O'Gara, F. & Condon, S. 1990 Thymidylate synthesis gene

from Lactococcus lactis as a genetic marker: An alternative to

antibiotic resistance genes. Applied & Environmental Microbiology

56, 2164±2169.

Ross, R.E., Keinath, A.P. & Cabota, Ma.A. 1998 Biological Control

of wirestem on cabbage using binucleate Rhizoctonia spp. Crop

Protection 17(2).

Saxena, A., Saxena, A. & Goel, R. 1998 Inter and intraspeci®c transfer

of antibiotic resistance from ¯uorexcent pseudomonads. Indian

Journal of Microbiology 38, 107±108.

Scanferlato, V.S., Orvos, D.R., Lacy, G.H. & Cairns, J.Jr. 1990

Enumerating low densities of genetically engineered Erwinia

carotovora in soil. Letters in Applied Microbiology 10, 55±59.

Seal, S.E., Cojackson, L.A. & Daniels, M.J. 1992 Use of tRNA

consensus primers to indicate subgroups of Pseudomonas solan-

acearum by polymerase chain reaction ampli®cation. Applied &


Shao, Z.Q. & Behki, R. 1996 Characterization of the expression of the

thCB gene, coding for a pesticide degrading cytochrome P-450 in

Rhodococcus strains. Applied and Environmental Microbiology

62, 403±407.

Shields, J.A., Paul, E.A., Lowe, W.E. & Parkinson, D. 1973 Turnover

of microbial tissue in soil under ®eld conditions. Soil Biology and

Biochemistry 5, 575±764.

Shim, J.S., Farrand, S.K. & Kerr, A. 1987 Biological control of crown

gall construction and testing of new biocontrol agents. Phytopath-

ology 77, 463±466.

Smiles, D.E. 1988 Aspects of the physical environment of soil

organisms. Biology of Fertility & Soil 6, 204±215.

Spreng, S. & Gentschev, I. 1998 Construction of chromosomally

encoded secreted hemolysin fusion proteins by use of mini-TnhlyA

(S) transposon. FEMS Microbiology Letters 165, 187±192.


Srivastava, J.S. 1990 Di�erential expression of pea symbiont plasmid.

PJBSJI in genetically dissimilar background. Microbios Letters

43, 135±140.

Srivastava, J.S. 1995 Occurrence and frequency of various micro¯ora

isolated from the rhizosphere of chickpea. Current Agricultural

Research 8, 82±85.

Ste�an, R.J. & Atlas, R.M. 1991 polymerase chain reaction: Appli-

cations in environmental microbiology. Annual Review of Micro-

biology 45, 137±161.

Svitel, J., Cru Curilla, I. & TKac, J. 1998 Microbial cell-based

biosensor for sensing glucose, sucrose or lactose. Biotechnology &

Applied Biochemistry 27, 153±158.

Trevors, J.T., Van Elsas, J.D., Van Overbeek, L.S. & Starodub, M.E.

1990 Transport of a genetically engineered Pseudomonas ¯uores-

cens strain through a soil microcosm. Applied & Environmental


Van der Bij, A.J., Dewager, W.J., Tucker. & Lutenberg, J.J. 1996

Plasmid stability in Pseudomonas ¯uorescens in the rhizosphere.

Applied & Environmental Microbiology 62, 1076±1080.

Van Elsas, J.D. & Trevors, J.T. 1990 Plasmid transfer to indigenors

bacteria in soil and rhizosphere: problems and perspectives. In

Bacterial genetics in Natural Environment. ed. Fry, J.C. & Day,

M.J. pp. 188±199. London: Chapman and Hall. ISBN 0-412-

35630-9.

Vincent, M.N., Harrison, L.A., Brackin, J.M., Kovacevich, R.,

Mukerji, P., Weller, D.M. & Pierson, E.A. 1991 Gentic analysis

of the antifungal activity of a soilborne Pseudomonas aureofaciens

strain. Applied & Environmental Microbiology 57, 2928±2934.

Walton, B.T. & Anderson, T.A. 1992. Plant, microprobe treatment

systems for toxic waste. Current Opinion in Biotechnology 3, 267±

428.

Way, J.L., Leong, P., Cannon, E., Morgan, R., Tamulinas, C., Way

J.L., Bazter, L., Nagi, A. & Chui, C. 1988 The mechanism of

cyanide introduction and its antegonism. In Cyanide Compounds in

Biology. ed. Evered, D. & Harnett, S. pp. 232±243. Chichester:

J. Wiley. ISBN 0-471-191904-7.

Weller, D.M. & Cook, R.J. 1983 Suppression of Take-all of wheat by

used treatments with ¯uorescent pseudomonas. Phytopathology

73, 463±469.

Williams, P., Bainton, J.J., Swift, S., Chhabra, S.R., Winson, M.K.,

Stewart, G.S.A.B., Salmond, G.P.C. & Bycroft, B.W. 1992

Small molecular mediated density-dependent control of gene

expression in prokaryotes: bioluminescence and the biosynthesis

of carbapenem antibiotics FEMS Microbiology Letters 100,

161±168.

Yurgel, S.N., Soberon, M., Sharypova L.A., Miranda, J., Morera, C.

& Simarov, B.V. 1998 Isolation of Sinorhizobium meliloti T5

mutants with altered cytochrome terminal oxidase expression and

improved symbiotic performance. FEMS Microbiology Letters

165, 167±173.


molecular and biochemical aspects of rhizobacterial ecology with emphasis on biological control

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