Directory UMM :Data Elmu:jurnal:A:Applied Soil Ecology:Vol15.Issue2.Oct2000:

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Endophytic communities of rhizobacteria and the strategies

required to create yield enhancing associations with crops

A.V. Sturz

a,∗

, J. Nowak

a_{Prince Edward Island Department of Agriculture and Forestry, PO Box 1600, Charlottetown, PEI, Canada C1A 7N3} b_{Department of Plant Science, Nova Scotia Agricultural College, Truro, NS, Canada B2N 5E3}

Received 31 May 1999; received in revised form 8 November 1999; accepted 23 March 2000

Abstract

The plant kingdom is colonized by a diverse array of endophytic bacteria which form non-pathogenic relationships with their hosts. When beneficial, such associations can stimulate plant growth, increase disease resistance, improve the plant’s ability to withstand environmental stresses (e.g. drought), or enhance N2fixation. Crop sequences can favour the build-up

of advantageous associations of bacterial endophyte populations leading to the development and maintenance of beneficial host-endophyte allelopathies. Utilization of rhizobacteria in sustainable crop production systems will require strategies to create and maintain beneficial bacterial populations within crops (endophytes) and as well in the soils surrounding those crops. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Beneficial association; Endophyte; PGPR; Sustainable crop production; Rhizosphere health

1. Introduction

Successive attempts to introduce beneficial bacte-ria into the rhizospheres of agricultural crops have generally met with varying degrees of failure due to the difficulties of incorporating non-resident bacterial components into established and acclimated micro-bial communities. For example, despite many years of attempting to modify naturally occurring soil pop-ulations of Rhizobium, such efforts have not been very successful (Brockwell et al., 1988; Thies et al., 1991).

Where candidate rhizobacteria have been intro-duced as biocontrol agents, their failure to control disease development has usually been attributed to

∗_{Corresponding author. Tel.:}₊_{1-902-368-5664;}

fax:+1-902-368-5661.

E-mail address: [email protected] (A.V. Sturz)

poor rhizosphere competence and the problems as-sociated with the instability of bacterial biocontrol agents in long-term culture (Schroth and Hancock, 1981; Weller, 1988). Consequently, root-associated bacteria as biological control agents have not yet be-come an established part of most pest management systems (Harman and Lumsden, 1990; Powell and Rhodes, 1994).

Considering the biodiversity of indigenous soil bacteria and the population densities involved, it is not surprising that it has proven difficult to make any long lasting structural changes to the composition of bacteria within any given soil-community. One strat-egy which may help contribute to the establishment of pre-selected beneficial organisms in root zone soils, and which has until recently been excluded from the research equation, is through fostering the early es-tablishment of selected communities of endophytic microorganisms within root systems.

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In recent times the term ‘endophyte’ has been ap-plied almost exclusively to fungi (Carroll, 1988; Clay, 1988); including the mycorrhizal fungi (O’Dell and Trappe, 1992). However, a more comprehensive defi-nition is one which includes ‘fungi or bacteria, which for all or part of their life cycle, invade the tissues of living plants and cause unapparent and asymptomatic infections entirely within plant tissues, but cause no symptoms of disease’ (Wilson, 1995).

The recovery of bacterial populations from the en-dodermis and root cortex of plants has been used to promote the idea that many bacteria in the rhi-zosphere are able to penetrate and colonize root tis-sues (Quadt-Hallman et al., 1997a,b). The inclusion of endophytic bacteria into the bacterial rhizosphere community was proposed by Darbyshire and Greaves (1973), and supported by Old and Nicolson (1978). In this model the root cortex becomes part of the soil–root microbial environment, resulting in a con-tinuous apoplastic pathway from the root epidermis to the shoot, sufficient for movement of microorganisms into the xylem (Petersen et al., 1981). Thus, a con-tinuum of root-associated microorganisms exist which are able to inhabit the rhizosphere, the root cortex and other plant organs (Kloepper et al., 1992).

2. Exo- versus endoroot bacteria

Conventional classifications, based on function, have grouped rhizobacteria — both those that exist outside (exoroot) and within root tissues (endoroot) — into two broad categories based on the relative benefit they confer to the plants with which they are associated. Thus, the deleterious rhizobacteria (DRB) (Fredrickson and Elliott, 1985; Schippers et al., 1987), are so-called because they are considered to adversely influence root health and plant well-being, while the plant growth promoting bacteria (PGPR) (see reviews by Glick, 1995; Arshad and Frankenberger, 1998) are considered to form part of a protective flora which provide benefit to the plant in the form of enhanced root function, disease suppression and accelerated plant development. The equivocal nature of such classifications has been pointed out by Nehl et al. (1996), as exoroot bacterial influence has been shown to fluctuate according to environmental conditions (Bakker et al., 1987; Chanway and Holl, 1994), host

genotype (Cherrington and Elliot, 1987; Åström and Gerhardson, 1988) and collateral mycorrhizal sta-tus (see reviews by Azcón-Aguilar and Barea, 1992; Linderman, 1994).

Interestingly, root health and cell longevity can be viewed as exclusive of rhizobacterial influence. Henry and Deacon (1981) proposed that, for most plants, rhi-zodermal and cortical cell death is an autolytic process which occurs in the absence of microorganism activity. Thus, the conventional view of root internal coloniza-tion by exoroot bacteria is one which occurs following rhizodermal autolysis (Darbyshire and Greaves, 1973; Foster and Rovira, 1978; Old and Nicolson, 1978). This led Foster and Bowen (1982) to consider that the population densities of organisms in the rhizoplane are the result of cell death and not its cause.

In all the above examples the emphasis has been on the influence of exoroot bacteria. However, plants can be colonized by a beneficial microbial endoflora prior to root autolysis (Frommel et al., 1991; Nowak, 1998). The specificity between endoroot bacteria and their hosts (Conn et al., 1997; Bensalim et al., 1998) is similar to that found in exoroot associations (Neal et al., 1970; Bowen and Rovira, 1976; Miller et al., 1989; Bolton et al., 1993; Merharg and Killham, 1995). van Peer et al. (1990) reported that endophytic and exoroot bacteria from the same genera formed discrete sub-populations each suited to colonizing their respective niches, and such adaptations do not appear to be easily reversible. McInroy and Kloepper (1995) observed that seed endophytes tend to develop into seedling endophytes. Bell et al. (1995), however, considered endophytic and rhizosphere populations of bacteria to be distinct, based on differences in their hydrolytic enzyme complement.

The community effect of endoroot bacteria on ex-oroot populations is seldom examined. However, the endoplant bacterial community can modify root mor-phology (Nowak, 1998) and ultrastructure (Benhamou et al., 1996) and may influence the way in which exoroot bacterial communities function and affect plant growth (Sturz, 1995; Sturz and Christie, 1995; Quadt-Hallman et al., 1997a). To paraphrase Andrews (1990), if we are to manipulate the assembled species and construct or enhance complimentary communi-ties of endo- and exorhizobacteria that confer positive benefits for crop production, a fuller appreciation of community structure and function, and the major

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organizing forces that govern such communities need to be determined.

3. A strategy for creating stable microbial communities

When considering the anthropogenic introduction of new-colonists (‘beneficial microorganisms’) into the root zone — through seed amendments or dur-ing seed-bed preparation — the potential for severe negative interactions with autochthonous microbial populations should be borne in mind (Atlas, 1986). It is now appreciated that the microbiological pop-ulations of an ecosystem are able to interact with one another through the production and reception of signalling molecules. Such signalling molecules can subsequently influence gene expression, and thereby bacterial phenotype (Salmond et al., 1995; Albus et al., 1997; Surette and Bassler, 1998). ‘Quo-rum sensing’ describes one such signalling system, whereby responses to bacterial population density are modulated through the accumulation of extracellular signalling molecules, that can regulate an assorted range of metabolic processes (Swift et al., 1996).

Similarly, the relationship between host and bacte-rial endophyte is not static. Communities of bactebacte-rial endophytes may not only be host specific, but also plant tissue sensitive, reacting and adapting at certain tissue sites and among certain tissue types within the host plant as it develops (Sturz et al., 1999). The dy-namic nature of bacterial phenotype expression, in this case antibiotic secretion, may be being governed by a phenomenon analogous to ‘quorum sensing’ — which can also be influenced by environmental factors such as oxygen concentration (Sitnikov et al., 1995).

While positive interactions (commensalism, mutu-alism, and synergism) may enable some populations to function as a community within a habitat (Rayner, 1997), negative interactions may result in the exclu-sion of microbial colonists from an established com-munity, or in a range of negative allelopathic events (Sturz and Christie, 1995, 1996).

In mature communities, positive interactions among autochthonous populations are usually better devel-oped than in newly established communities. The successful establishment of beneficial organisms will be influenced, to varying degrees, by the network

of connections among species in a mature (estab-lished) ecosystem. In essence, the establishment of the ‘new-colonist’ population can be prejudiced by the dynamics of the ecosystem it is trying to invade, through a form of defensive mutualism (Clay, 1988).

Thus, one component of an approach designed to favour the successful assimilation of selected organ-isms into a rhizosphere, would be to introduce the ben-eficial microorganism(s) at the earliest possible stage in the metapopulation continuum (Levins, 1976; Hast-ings and Harrison, 1994). As endophytic bacteria have been recovered from the ovules, seeds and tubers of a variety of plants (Mundt and Hinkle, 1976; Holland and Polacco, 1994), the creation of selected commu-nities of beneficial bacterial endophytes within these germinal structures would form one of the earliest pio-neer colonization events possible. Initially, such com-munities may be relatively stable and could compete with native soil bacteria once plant propagules had been planted.

4. Engineering microbial communities

The ability to successfully manipulate endophytic bacteria in agricultural production systems will depend upon the ability to select, incorporate and maintain beneficial microbial populations in the field. How-ever, the reciprocity among populations of exo- and endorhizal origin has not been fully explored. If the composition and function of endophytic populations is determined by co-existing rhizosphere populations, then altering the exoroot community may be unde-sirable; especially where associations of co-operating species occupy a single niche that could not be col-onized by either partner population alone (Henry, 1966). A number of strategies may enable the early establishment of selected beneficial microbial popula-tions within the host and in the surrounding field soil. Biotization. One of the more elegant ways to in-troduce selections of endophytes into the host plant, at an early stage, would be through tissue culture (Varga et al., 1994; Nowak, 1998). Biotization, in the current context, may be defined as the metabolic re-sponse of in vitro grown plant material to microbial inoculant(s) which promote developmental and physi-ological changes that enhance biotic and abiotic stress resistance in subsequent plant progeny. Such systems

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allow for mutual adaptation between the host plant and the introduced bacteria (Nowak et al., 1999; Sturz and Nowak, unpublished data). The benefits of an established, thriving and stable microbial endoplant community can include disease resistance, through the de novo synthesis of structural compounds and fungitoxic metabolites at sites of attempted fungal penetration (Benhamou et al., 1996), the induction and expression of general molecular-based plant im-munity (Richards, 1997; Sticher et al., 1997; Nowak et al., 1998), or the simple exclusion of other organisms (phytopathogens or colonists) by niche competition. Bacterized plantlets not only grow faster than un-bacterized plantlets (Chanway, 1997; Bensalim et al., 1998), but they are sturdier, have a better developed root system (Nowak, 1998) and a significantly greater capacity to withstand adverse biotic stresses (i.e., drought) and low level disease pressures (Stewart, 1997; Sharma and Nowak, 1998). In potato culture, endophyte bacteria can be translocated to successive generations of potato plants during multiplication, either through stem explants (Frommel et al., 1991), microtubers (Nowak and Sturz, unpublished) or in seeds (Varga, personal communication). Of recent in-terest to sustainable agriculture systems has been the realization that stable, beneficial associations between plant species and diazotrophic bacteria (Varga et al., 1994; Preininger et al., 1997) under conditions of low soil nitrogen (Gyurján et al., 1995) may be used to improve plant growth and crop productivity.

Crop production systems. Crop rotations and tillage management have been shown to influence specific soil microbial populations (see reviews by Alabou-vette et al., 1996; Sturz et al., 1997). Selecting crop production systems which sustain and encourage the development of consortia of beneficial rhizobacterial populations will be crucial, if the cumulative bene-fits of microbial synergies are to be harnessed. It is likely that such benefits will be small in any given season, and their incremental value only recognized over time. In this respect, the iatrogenic effects be-tween agrichemicals and non-target exo- and endo-root microflora bears closer examination (Ingham, 1985; Bollen, 1993), as long-term applications of crop protection chemicals may adversely affect soil fertility by reducing the quantity and quality of bene-ficial rhizobacteria populations (Sturz and Kimpinski, 1999).

Cultivar selection. It is generally acknowledged that rhizobacterial populations can be manipulated, in the short term, through plant species selection (Neal et al., 1970; Grayston et al., 1998). Root exudates can determine, to a great extent, which organisms will re-side in the rhizoplane (Cook and Baker, 1983; Kunc and Macura, 1988). Rhizobacteria can, themselves, spur a root exudation response in plants (Bowen and Rovira, 1976; Bolton et al., 1993) that is species spe-cific (Chanway et al., 1988; Merharg and Killham, 1995). Such close interactions have prompted specu-lation that rhizobacteria and plants have co-evolved; plants encouraging the establishment of specific and beneficial rhizospheres through the selective exuda-tion of specific root exudates (Bolton et al., 1993).

This close relationship between plants and rhizobac-teria is also found to extend to endophytic bacrhizobac-teria. In some cases complementary crops grown in rotation can share 70% of the same species of endophytic bac-teria (Sturz et al., 1998). Such associations between different crop species can be cultivar specific. Thus, certain cultivars of clover can foster the development of rhizo- and endophytic bacteria which favour the growth and development of specific cultivars of pota-toes (Sturz and Christie, 1998).

Genetic modification. Altering the genetic make-up of plants to manipulate both internal and external bacterial populations offers the possibility of creat-ing preferred rhizosphere communities (O’Connell et al., 1996). Other than research into rhizobia–legume interactions, most selection criteria in plant breeding programs have not considered which component(s) of superior progeny performance are attributable to the inherited ability of plants to respond to, modify or create communities of beneficial bacteria in their rhizospheres. Even so, it is likely that there has been some collateral selection for host-endophyte interac-tive ability.

To capitalize further on such associations, breeding programs could proceed in a number of directions. Im-proved plant performance, based on superior interac-tions between host plants and their endophytes, could result in yield benefits; either directly, or indirectly through a healthier, vigorous and more stress resistant crop. Alternatively, selections could be based upon host responsiveness to specific beneficial bacteria, which would then become a part of any bacterization step during multiplication, e.g. interactions between

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temperature, bacterization and potato genotype indi-cate the importance of clonal variations for utilization of beneficial microorganisms in potato production under heat stress conditions (Bensalim et al., 1998).

Several strategies have already been proposed to optimize endophyte nitrogen fixation in non-legume crops, including: (i) altering the receptivity of the host plant to colonization by nitrogen-fixing bacte-ria through nodule induction (de Bruijn et al., 1995; Christiansen-Weniger, 1998); (ii) exploiting stable plant–diazotrophic endophyte bacteria associations able to fix nitrogen endophytically (Reddy and Ladha, 1995; Kennedy et al., 1997; Stoltzfus et al., 1997; Swensen and Mullin, 1997) and (iii) through the ge-netic alteration of selected endophytic bacteria, or direct incorporation of nitrogen-fixing genes (Dixon et al., 1997; Gough et al., 1997). The reader is referred to reviews in Ladha et al. (1997).

Seed treatments. Judging by past experience, ap-plying bacterial seed treatments prior to planting does not guarantee the establishment of a beneficial endo-or exendo-orhizal flendo-ora (Frommel et al., 1993) nendo-or does it always enhance yield (Volkmar and Bremer, 1998). Introductions of non-local microfloras must compete with established microbial communities in the soil, the rhizosphere and within the plant. Both true seeds and plants which are propagated vegetatively are likely to carry enduring consortia of adapted endo-phytes, a portion of which will be transferred to the subsequent progeny. Niche specialization will ensure that local communities are better positioned to col-onize and retain niche dominance at the expense of later introduced species. Our feeling, at the present time, is that seed treatments are best suited to aug-menting established consortia of microbial organisms (fungal, bacterial and mycorrhizal) created as part of a long-term strategy of harmonized crop (cultivar) selection and management practices.

5. Conclusions

Current interest in beneficial rhizobacteria has fo-cused on the exoroot and its associated rhizosphere community. However, plants are also colonized by a diverse array of endophytic bacteria which form non-pathogenic relationships within plants. Positive interactions between endophytes and their host plants

can result in a range of beneficial effects which are similar if not complementary with those reported for the exorhizobacteria. These include increased plant growth and development, resistance to disease and improvements in the host plant’s ability to withstand environmental stresses (e.g. drought). Endophytes offer the twin benefits of being acclimated to their hosts, and present at seedling development and rhi-zosphere initiation. These factors provide endophytes with a competitive ecological advantage compared to the resident ‘wild-type’ soil microflora that are so often implicated in the failure of biological seed treatments (biocontrol agents and growth promotion amendments). However, much of the basic informa-tion regarding endophyte community structure, their principal functions, relative ecological stability, and the organizing forces that govern their continuity, is still lacking. If rhizobacteria are to be better utilized in crop production systems, then one approach to enable this to happen should involve the creation and enhancement of sustainable, beneficial communities of bacteria in the endo- as well as the exoroot.

References

Alabouvette, C., Hoeper, H., Lemanceau, P., Steinberg, C., 1996. Soil suppressiveness to diseases induced by soilborne plant pathogens. In: Stotzky, G., Bollag, J.-M. (Eds.), Soil Biochemistry, Vol. 9. Dekker, New York, pp. 371–413. Albus, A.M., Pesci, E.C., Runyen-Janecky, L.J., West, S.E.H.,

Iglewski, B.H., 1997. Vfr controls quorum sensing in

Pseudomonas aeruginosa. J. Bacteriol. 179, 3928–3935.

Andrews, J.H., 1990. Biological control in the phyllosphere: realistic goal or false hope? Can. J. Plant Pathol. 12, 300–307. Arshad, M., Frankenberger Jr., W.T., 1998. Plant growth substances in the rhizosphere: microbial production and functions. Adv. Agron. 62, 46–151.

Åström, B., Gerhardson, B., 1988. Differential reactions of wheat and pea genotypes to root inoculation with growth-affecting rhizosphere bacteria. Plant Soil 109, 263–269.

Atlas, R.M., 1986. Applicability of general ecological principles to microbial ecology. In: Poindexter, J.S., Leadbetter, E.R. (Eds.), Bacteria in Nature, Vol. 2. Plenum Press, New York, pp. 339–370.

Azcón-Aguilar, C., Barea, J.M., 1992. Interactions between mycorrhizal fungi and other rhizosphere microorganisms. In: Allen, M.F. (Ed.), Mycorrhizal Functioning: An Integrative Plant–Fungal Process. Chapman & Hall, New York, pp. 163–198.

Bakker, P.A.H.M., Bakker, A.W., Marugg, J.D., Weisbeck, P.J., Schippers, B., 1987. Bioassay for studying the role of

(6)

siderophores in potato stimulation by Pseudomonas spp. in short potato rotations. Soil Biol. Biochem. 19, 443–449. Bell, C.R., Dickie, G.A., Harvey, W.L.G., Chan, J.W.Y.F., 1995.

Endophytic bacteria in grapevine. Can. J. Microbiol. 41, 46–53. Benhamou, N., Kloepper, J.W., Quadt-Hallman, A., Tuzun, S., 1996. Induction of defence-related ultrastructural modifications in pea root tissues inoculated with endophytic bacteria. Plant Physiol. 112, 919–929.

Bensalim, S., Nowak, J., Asiedu, S., 1998. A plant growth promoting rhizobacterium and temperature effects on perfor-mance of 18 clones of potato. Am. Pot. J. 75, 145–152. Bollen, G.J., 1993. Mechanisms involved in nontarget effects

of pesticides on soil-borne pathogens. In: Altman, J. (Ed.), Pesticide Interactions in Crop Production: Beneficial and Deleterious Effects. CRC Press, Boca Raton, FL, pp. 281–301. Bolton, H.J., Fredrickson, J.K., Elliott, L.F., 1993. Microbial ecology of the rhizosphere. In: Metting, F.B.J. (Ed.), Soil Microbial Ecology. Dekker, New York, pp. 27–63.

Bowen, G.D., Rovira, A.D., 1976. Microbial colonization of plant roots. Ann. Rev. Phytopathol. 14, 121–144.

Brockwell, J., Herridge, D.F., Morthorpe, L.J., Roughley, R.J., 1988. Numerical effects of Rhizobium population on legume symbiosis. In: Beck, D.P., Materon, L.A. (Eds.), Nitrogen Fixation by Legumes in Mediterranean Agriculture. Marunus Nijhoff, Dordrecht, pp. 178–193.

Carroll, G., 1988. Fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbiont. Ecology 69, 2–9. Chanway, C.P., 1997. Inoculation of tree roots with plant

growth promoting soil bacteria: an emerging technology for reforestation. For. Sci. 43, 99–112.

Chanway, C.P., Holl, F.B., 1994. Growth of outplanted lodgepole pine seedlings one year after inoculation with growth promoting rhizobacteria. For. Sci. 40, 238–246.

Chanway, C.P., Nelson, L.M., Holl, F.B., 1988. Cultivar-specific growth promotion of spring wheat (Triticum aestivum L.) by coexistent Bacillus species. Can. J. Microbiol. 34, 925–929. Cherrington, C.A., Elliot, L.F., 1987. Incidence of inhibitory

pseudomonads in the Pacific Northwest. Plant Soil 101, 159– 165.

Christiansen-Weniger, C., 1998. Endophytic establishment of diazotrophic bacteria in auxin induced tumours of cereal crops. CRC Crit. Rev. Plant Sci. 17, 55–76.

Clay, K., 1988. Fungal endophytes of grasses: a defensive mutualism between plants and fungi. Ecology 69, 10–16. Conn, K.L., Nowak, J., Lazarovits, G., 1997. A gnotobiotic

bioassay for studying interactions between potatoes and plant growth-promoting rhizobacteria. Can. J. Microbiol. 43, 801– 808.

Cook, R.J., Baker, K.F., 1983. The Nature and Practice of Biological Control of Plant Pathogens. American Phytopatho-logical Society, St. Paul, MN.

Darbyshire, J.F., Greaves, M.P., 1973. Bacteria and protozoa in the rhizosphere. Pestic. Sci. 4, 349–360.

de Bruijn, F.J., Jing, Y., Dazzo, F.B., 1995. Potential pitfalls of trying to extend symbiotic interactions of nitrogen-fixing organisms to presently non-nodulated plants, such as rice. Plant Soil 174, 225–240.

Dixon, R., Cheng, Q., Shen, G.-F., Day, A., Dowson-Day, M., 1997. Nif gene transfer and expression in chloroplasts: prospects and problems. Plant Soil 194, 193–203.

Foster, R.C., Bowen, G.D., 1982. Plant surfaces and bacterial growth: the rhizosphere and rhizoplane. In: Mount, M.S., Lacy, G.H. (Eds.), Phytopathogenic Prokaryotes. Dekker, New York, pp. 159–185.

Foster, R.C., Rovira, A.D., 1978. Ultrastructure of the wheat rhizosphere. New Phytol. 76, 343–352.

Fredrickson, J.K., Elliott, L.F., 1985. Effects on winter wheat seedling growth by toxin producing rhizobacteria. Plant Soil 83, 399–409.

Frommel, M.I., Nowak, J., Lazarovits, G., 1991. Growth enhancement and developmental modifications of in vitro grown potato (Solanum tuberosum ssp. tuberosum) as affected by a nonfluorescent Pseudomonas sp. Plant Physiol. 96, 928–936. Frommel, M.I., Nowak, J., Lazarovits, G., 1993. Treatment of

potato tubers with a growth promoting Pseudomonas sp.: bacterium distribution in the rhizosphere and plant growth responses. Plant Soil 150, 51–60.

Glick, B.R., 1995. The enhancement of plant growth by free-living bacteria. Can. J. Microbiol. 41, 109–117.

Gough, C., Vasse, J., Galera, C., Webster, G., Cocking, E., Dénarié, J., 1997. Interactions between bacterial diazotrophs and non-legume dicots: Arabidopsis thaliana as a model plant. Plant Soil 194, 123–130.

Grayston, S.J., Wang, S., Campbell, C.D., Edwards, A.C., 1998. Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biol. Biochem. 30, 369–378.

Gyurján, I., Korányi, P., Preininger, É., Varga, S.S., Paless, G., 1995. Artificial plant–Azotobacter symbiosis for atmospheric nitrogen fixation. In: Fendrik, I. (Ed.), Azospirillum VI and Related Microorganisms. NATO ASI Series, Vol. 37. Springer, Berlin, pp. 401–413.

Harman, G.E., Lumsden, R.D., 1990. Biological disease control. In: Lynch, J.M. (Ed.), The Rhizosphere. Wiley, London, pp. 259–280.

Hastings, A., Harrison, S., 1994. Metapopulation dynamics and genetics. Ann. Rev. Ecol. Syst. 25, 167–188.

Henry, S.M., 1966. Symbiosis. Academic Press, New York. Henry, C.M., Deacon, J.W., 1981. Natural (nonpathogenic) death

of the cortex of wheat and barley seminal roots, as evidenced by nuclear staining with acridine orange. Plant Soil 60, 255–274. Holland, M.A., Polacco, J.C., 1994. PPFM’s and other covert contaminants: is there more to plant physiology than just plant? Ann. Rev. Plant Physiol. Plant Mol. Biol. 45, 197–209. Ingham, E.R., 1985. Review of the effects of twelve selected

biocides on target and non-target soil organisms. Crop Protection 4, 3–32.

Kennedy, I.R., Pereg-Gerk, L.L., Wood, C., Deaker, R., Gilcrest, K., Katupitiya, S., 1997. Biological nitrogen fixation in non-leguminous field crops: facilitating the evolution of an effective association between Azospirillum and wheat. Plant Soil 194, 65–79.

Kloepper, J.W., Schippers, B., Bakker, P.A.H.M., 1992. Proposed elimination of the term endorhizosphere. Phytopathology 82, 726–727.

(7)

Kunc, F., Macura, J., 1988. Mechanisms of adaptation and selection of microorganisms in the soil. In: Vancura, V., Kunc, F. (Eds.), Soil Microbial Associations. Elsevier, Amsterdam, pp. 281–299. Ladha, J.K., de Bruijn, F.J., Malik, K.A., 1997. Opportunities for biological nitrogen fixation in rice and other non-legumes. In: Ladha, J.K., de Bruijn, F.J., Malik, K.A. (Eds.), Papers Presented at the Second Working Group of the Frontier Project on Nitrogen Fixation in Rice, NIBGE, Faisalabad, Pakistan, October 13–15, 1996. Plant Soil 194 (1/2).

Levins, R., 1976. Extinction. Lectures Math. Life Sci. 2, 75–107. Linderman, R.G., 1994. Role of VAM in biocontrol. In: Pfleger, F.L., Linderman, R.G. (Eds.), Mycorrhizae and Plant Health. American Phytopathological Society, St. Paul, MN, pp. 1–25. McInroy, J.A., Kloepper, J.W., 1995. Survey of indigenous bacterial

endophytes from cotton and sweet corn. Plant Soil 173, 337– 342.

Merharg, A.A., Killham, K., 1995. Loss of exudates from roots of perennial rye grass inoculated with a range of microorganisms. Plant Soil 170, 345–349.

Miller, H.J., Hencken, G., Van Veen, J.A., 1989. Variation and composition in the rhizospheres of maize, wheat and grass cultivars, wheat and grass cultivars. Can. J. Microbiol. 35, 656– 660.

Mundt, J.O., Hinkle, N.F., 1976. Bacteria within ovules and seeds. Appl. Environ. Microbiol. 32, 694–698.

Neal, J.L., Atkinson, T.G., Larson, R.I., 1970. Changes in the rhizosphere microflora of spring wheat induced by disomic substitution of a chromosome. Can. J. Microbiol. 16, 153–158. Nehl, D.B., Allen, S.J., Brown, J.F., 1996. Deleterious rhizosphere bacteria: an integrating perspective. Appl. Soil Ecol. 5, 1–20. Nowak, J., 1998. Benefits of in vitro “biotization” of plant tissue

cultures with microbial inoculants. In Vitro Cell. Dev. Biol. Plant 34, 122–130.

Nowak, J., Asiedu, S.K., Bensalim, S., Richards, J., Stewart, A., Smith, C., Stevens, D., Sturz, A.V., 1998. From laboratory to applications: challenges and progress with in vitro dual cultures of potato and beneficial bacteria. Plant Cell Tiss. Organ. Cult. 52, 97–103.

Nowak, J., Bensalim, S., Smith, C.D., Dunbar, C., Asiedu, S.K., Madani, A., Lazarovits, G., Northcott, D., Sturz, A.V., 2000. Microplantlet weaning by in vitro bacterization. Potato Res., in press.

O’Connell, K.P., Goodman, R.M., Handelson, J., 1996. Engineer-ing the rhizosphere: expressEngineer-ing a bias. Trends-biotechnology 14, 83–88.

O’Dell, T.E., Trappe, J.M., 1992. Root endophytes of lupin and some other legumes in northwestern USA. New Phytol. 122, 479–485.

Old, K.M., Nicolson, T.H., 1978. The root cortex as part of a microbial continuum. In: Loutit, M.V., Miles, J.A.R. (Eds.), Microbial Ecology. Springer, Berlin, pp. 291–294.

Petersen, C.A., Emanuel, M.E., Humphreys, G.B., 1981. Pathway of movement of apoplastic fluorescent dye tracers through the endodermis at the site of secondary root formation in corn (Zea

mays) and broad bean (Vicia faba). Can. J. Bot. 59, 618–625.

Powell, K.A., Rhodes, D.J., 1994. Strategies for the progression of biological fungicides into field evaluation. BCPC Monograph No. 59, pp. 307–315.

Preininger, É., Zatyko, J., Szucs, P., Korányi, P., Gyurján, I., 1997. In vitro establishment of nitrogen-fixing strawberry (Fragaria×annassa) via artificial symbiosis with Azomonas insignis. In Vitro Cell. Dev. Biol. Plant 33, 190–194.

Quadt-Hallman, A., Hallman, J., Kloepper, J.W., 1997a. Bacterial endophytes in cotton: location and interaction with other plant associated bacteria. Can. J. Microbiol. 43, 254–259.

Quadt-Hallman, A., Benhamou, N., Kloepper, J.W., 1997b. Bacterial endophytes in cotton: mechanisms of entering the plant. Can. J. Microbiol. 43, 577–582.

Rayner, A.D.M., 1997. Degrees of Freedom: Living in Dynamic Boundaries. Imperial College Press, London, 312 pp. Reddy, P.M., Ladha, J.K., 1995. Can symbiotic nitrogen fixation

be extended to rice? Nitrogen fixation fundamentals and applications. In: Proceedings of the International Congress on Nitrogen Fixation, Vol. 27. St. Petersburg, Russia, May 28–June 3, 1995, pp. 629–633.

Richards, J., 1997. Induced resistance responses in potato inoculated in vitro with a plant growth promoting pseudomonad. M.Sc. Thesis. Dalhousie University, Halifax, NS, Canada. Salmond, G.P.C., Bycroft, B.W., Stewart, G.S.A.B., Williams, P.,

1995. The bacterial ‘enigma’: cracking the code of cell–cell communication. Mol. Microbiol. 16, 615–624.

Schippers, B., Bakker, A.W., Bakker, P.A.H.M., 1987. Interactions of deleterious and beneficial rhizosphere microorganisms and the effect of cropping practices. Ann. Rev. Phytopathol. 25, 339–358.

Schroth, M.N., Hancock, J.G., 1981. Selected topics in biological control. Ann. Rev. Microbiol. 35, 453–476.

Sharma, V.K., Nowak, J., 1998. Enhancement of verticillium wilt resistance in tomato transplants by in vitro co-culture of seedlings with a plant growth promoting rhizobacterium (Pseudomonas sp. strain. PsJN). Can. J. Microbiol. 44, 528– 536.

Sitnikov, D.M., Schineller, J.B., Baldwin, T.O., 1995. Trans-criptional regulation of bioluminescence genes from Vibrio

fischeri. Mol. Microbiol. 17, 801–812.

Stewart, A.H., 1997. Suppression of verticillium wilt in potatoes with a plant growth promoting rhizobacterium. M.Sc. Thesis. Dalhousie University, Halifax, NS, Canada.

Sticher, L., Mauch-Mani, B., Métraux, J.P., 1997. Systemic acquired resistance. Ann. Rev. Phytopathol. 35, 235–270. Stoltzfus, J.R., So, R., Malarvithi, P.P., Ladha, J.K., 1997. Isolation

of endophytic bacteria from rice and assessment of their potential for supplying rice with biologically fixed nitrogen. Plant Soil 194, 25–36.

Sturz, A.V., 1995. The role of endophytic bacteria during seed piece decay and potato tuberization. Plant Soil 175, 257–263. Sturz, A.V., Christie, B.R., 1995. Endophytic bacterial systems governing red clover growth and development. Ann. Appl. Biol. 126, 285–290.

Sturz, A.V., Christie, B.R., 1996. Endophytic bacteria of red clover as causal agents of allelopathic clover–maize syndromes. Soil Biol. Biochem. 28, 583–588.

Sturz, A.V., Christie, B.R., 1998. The potential benefits from cultivar specific red clover–potato crop rotations. Ann. Appl. Biol. 133, 365–373.

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Sturz, A.V., Kimpinski, J., 1999. The influence of fosthiazate and aldicarb on populations of plant growth promoting bacteria and bacterial feeding nematodes in the potato root zone. Plant Pathol. 48, 26–32.

Sturz, A.V., Carter, M.R., Johnston, H.W., 1997. A review of plant disease, pathogen interactions and microbial antagonism under conservation tillage in temperate humid agriculture. Soil Till. Res. 41, 169–189.

Sturz, A.V., Christie, B.R., Matheson, B.G., 1998. Associations of bacterial endophyte populations from red clover and potato crops with potential for beneficial allelopathy. Can. J. Microbiol. 44, 162–167.

Sturz, A.V., Christie, B.R., Matheson, B.G., Arsenault, W.J., Buchanan, N.A., 1999. Endophytic bacterial communities in the periderm of potato tubers and their potential to improve resistance to soil-borne plant pathogens. Plant Pathol. 48, 360– 370.

Surette, M.G., Bassler, B.L., 1998. Quorum sensing in Escherichia

coli and Salmonella typhimurium. Proc. Natl. Acad. Sci. USA

95, 7046–7050.

Swensen, S.M., Mullin, B.C., 1997. The impact of molecular systematics on the hypotheses for the evolution of root nodule symbioses and implications for expanding symbioses to new host plant genera. Plant Soil 194, 185–192.

Swift, S., Throup, J.P., Williams, P., Salmon, D.G.P.C., Stewart, G.S.A.B., 1996. Quorum sensing: a population density component in the determination of bacterial phenotype. Trends-biochem. Sci. 21, 214–219.

Thies, J.E., Singleton, P.W., Bohlool, B.B., 1991. Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field grown legumes. Appl. Environ. Microbiol. 57, 19–28.

van Peer, R., Punte, H.L.M., De Weger, L.A., Schippers, B., 1990. Characterization of root surface and endorhizosphere pseudomonads in relation to their colonization of roots. Appl. Environ. Microbiol. 56, 2462–2470.

Varga, S.S., Korányi, P.A., Preininger, É., Gyrurán, I., 1994. Artificial associations between Daucus and nitrogen-fixing

Azotobacter cells in vitro. Physiol. Plant. 90, 789–790.

Volkmar, K.M., Bremer, E., 1998. Effects of seed inoculation with a strain of Pseudomonas fluorescens on root growth and activity of wheat in well-watered and drought stressed glass-fronted rhizotrons. Can. J. Plant Sci. 78, 545–551.

Weller, D.M., 1988. Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Ann. Rev. Phytopathol. 26, 379–407.

Wilson, D., 1995. Endophyte — the evolution of a term, and clarification of its use and definition. Oikos 73, 274–276.

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organizing forces that govern such communities need

to be determined.

3. A strategy for creating stable microbial

communities

When considering the anthropogenic introduction

of new-colonists (‘beneficial microorganisms’) into

the root zone — through seed amendments or

dur-ing seed-bed preparation — the potential for severe

negative interactions with autochthonous microbial

populations should be borne in mind (Atlas, 1986).

It is now appreciated that the microbiological

pop-ulations of an ecosystem are able to interact with

one another through the production and reception

of signalling molecules. Such signalling molecules

can subsequently influence gene expression, and

thereby bacterial phenotype (Salmond et al., 1995;

Albus et al., 1997; Surette and Bassler, 1998).

‘Quo-rum sensing’ describes one such signalling system,

whereby responses to bacterial population density are

modulated through the accumulation of extracellular

signalling molecules, that can regulate an assorted

range of metabolic processes (Swift et al., 1996).

Similarly, the relationship between host and

bacte-rial endophyte is not static. Communities of bactebacte-rial

endophytes may not only be host specific, but also

plant tissue sensitive, reacting and adapting at certain

tissue sites and among certain tissue types within the

host plant as it develops (Sturz et al., 1999). The

dy-namic nature of bacterial phenotype expression, in this

case antibiotic secretion, may be being governed by a

phenomenon analogous to ‘quorum sensing’ — which

can also be influenced by environmental factors such

as oxygen concentration (Sitnikov et al., 1995).

While positive interactions (commensalism,

mutu-alism, and synergism) may enable some populations

to function as a community within a habitat (Rayner,

1997), negative interactions may result in the

exclu-sion of microbial colonists from an established

com-munity, or in a range of negative allelopathic events

(Sturz and Christie, 1995, 1996).

In mature communities, positive interactions among

autochthonous populations are usually better

devel-oped than in newly established communities. The

successful establishment of beneficial organisms will

be influenced, to varying degrees, by the network

of connections among species in a mature

(estab-lished) ecosystem. In essence, the establishment of

the ‘new-colonist’ population can be prejudiced by

the dynamics of the ecosystem it is trying to invade,

through a form of defensive mutualism (Clay, 1988).

Thus, one component of an approach designed to

favour the successful assimilation of selected

organ-isms into a rhizosphere, would be to introduce the

ben-eficial microorganism(s) at the earliest possible stage

in the metapopulation continuum (Levins, 1976;

Hast-ings and Harrison, 1994). As endophytic bacteria have

been recovered from the ovules, seeds and tubers of

a variety of plants (Mundt and Hinkle, 1976; Holland

and Polacco, 1994), the creation of selected

commu-nities of beneficial bacterial endophytes within these

germinal structures would form one of the earliest

pio-neer colonization events possible. Initially, such

com-munities may be relatively stable and could compete

with native soil bacteria once plant propagules had

been planted.

4. Engineering microbial communities

The ability to successfully manipulate endophytic

bacteria in agricultural production systems will depend

upon the ability to select, incorporate and maintain

beneficial microbial populations in the field.

How-ever, the reciprocity among populations of exo- and

endorhizal origin has not been fully explored. If the

composition and function of endophytic populations

is determined by co-existing rhizosphere populations,

then altering the exoroot community may be

unde-sirable; especially where associations of co-operating

species occupy a single niche that could not be

col-onized by either partner population alone (Henry,

1966). A number of strategies may enable the early

establishment of selected beneficial microbial

popula-tions within the host and in the surrounding field soil.

Biotization. One of the more elegant ways to

in-troduce selections of endophytes into the host plant,

at an early stage, would be through tissue culture

(Varga et al., 1994; Nowak, 1998). Biotization, in the

current context, may be defined as the metabolic

re-sponse of in vitro grown plant material to microbial

inoculant(s) which promote developmental and

physi-ological changes that enhance biotic and abiotic stress

resistance in subsequent plant progeny. Such systems

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allow for mutual adaptation between the host plant

and the introduced bacteria (Nowak et al., 1999; Sturz

and Nowak, unpublished data). The benefits of an

established, thriving and stable microbial endoplant

community can include disease resistance, through

the de novo synthesis of structural compounds and

fungitoxic metabolites at sites of attempted fungal

penetration (Benhamou et al., 1996), the induction

and expression of general molecular-based plant

im-munity (Richards, 1997; Sticher et al., 1997; Nowak et

al., 1998), or the simple exclusion of other organisms

(phytopathogens or colonists) by niche competition.

Bacterized plantlets not only grow faster than

un-bacterized plantlets (Chanway, 1997; Bensalim et al.,

1998), but they are sturdier, have a better developed

root system (Nowak, 1998) and a significantly greater

capacity to withstand adverse biotic stresses (i.e.,

drought) and low level disease pressures (Stewart,

1997; Sharma and Nowak, 1998). In potato culture,

endophyte bacteria can be translocated to successive

generations of potato plants during multiplication,

either through stem explants (Frommel et al., 1991),

microtubers (Nowak and Sturz, unpublished) or in

seeds (Varga, personal communication). Of recent

in-terest to sustainable agriculture systems has been the

realization that stable, beneficial associations between

plant species and diazotrophic bacteria (Varga et al.,

1994; Preininger et al., 1997) under conditions of low

soil nitrogen (Gyurján et al., 1995) may be used to

improve plant growth and crop productivity.

Crop production systems. Crop rotations and tillage

management have been shown to influence specific

soil microbial populations (see reviews by

Alabou-vette et al., 1996; Sturz et al., 1997). Selecting crop

production systems which sustain and encourage the

development of consortia of beneficial rhizobacterial

populations will be crucial, if the cumulative

bene-fits of microbial synergies are to be harnessed. It is

likely that such benefits will be small in any given

season, and their incremental value only recognized

over time. In this respect, the iatrogenic effects

be-tween agrichemicals and non-target exo- and

endo-root microflora bears closer examination (Ingham,

1985; Bollen, 1993), as long-term applications of

crop protection chemicals may adversely affect soil

fertility by reducing the quantity and quality of

bene-ficial rhizobacteria populations (Sturz and Kimpinski,

1999).

Cultivar selection. It is generally acknowledged that

rhizobacterial populations can be manipulated, in the

short term, through plant species selection (Neal et

al., 1970; Grayston et al., 1998). Root exudates can

determine, to a great extent, which organisms will

re-side in the rhizoplane (Cook and Baker, 1983; Kunc

and Macura, 1988). Rhizobacteria can, themselves,

spur a root exudation response in plants (Bowen and

Rovira, 1976; Bolton et al., 1993) that is species

spe-cific (Chanway et al., 1988; Merharg and Killham,

1995). Such close interactions have prompted

specu-lation that rhizobacteria and plants have co-evolved;

plants encouraging the establishment of specific and

beneficial rhizospheres through the selective

exuda-tion of specific root exudates (Bolton et al., 1993).

This close relationship between plants and

rhizobac-teria is also found to extend to endophytic bacrhizobac-teria.

In some cases complementary crops grown in rotation

can share 70% of the same species of endophytic

bac-teria (Sturz et al., 1998). Such associations between

different crop species can be cultivar specific. Thus,

certain cultivars of clover can foster the development

of rhizo- and endophytic bacteria which favour the

growth and development of specific cultivars of

pota-toes (Sturz and Christie, 1998).

Genetic modification. Altering the genetic make-up

of plants to manipulate both internal and external

bacterial populations offers the possibility of

creat-ing preferred rhizosphere communities (O’Connell et

al., 1996). Other than research into rhizobia–legume

interactions, most selection criteria in plant breeding

programs have not considered which component(s)

of superior progeny performance are attributable to

the inherited ability of plants to respond to, modify

or create communities of beneficial bacteria in their

rhizospheres. Even so, it is likely that there has been

some collateral selection for host-endophyte

interac-tive ability.

To capitalize further on such associations, breeding

programs could proceed in a number of directions.

Im-proved plant performance, based on superior

interac-tions between host plants and their endophytes, could

result in yield benefits; either directly, or indirectly

through a healthier, vigorous and more stress resistant

crop. Alternatively, selections could be based upon

host responsiveness to specific beneficial bacteria,

which would then become a part of any bacterization

step during multiplication, e.g. interactions between

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temperature, bacterization and potato genotype

indi-cate the importance of clonal variations for utilization

of beneficial microorganisms in potato production

under heat stress conditions (Bensalim et al., 1998).

Several strategies have already been proposed to

optimize endophyte nitrogen fixation in non-legume

crops, including: (i) altering the receptivity of the

host plant to colonization by nitrogen-fixing

bacte-ria through nodule induction (de Bruijn et al., 1995;

Christiansen-Weniger, 1998); (ii) exploiting stable

plant–diazotrophic endophyte bacteria associations

able to fix nitrogen endophytically (Reddy and Ladha,

1995; Kennedy et al., 1997; Stoltzfus et al., 1997;

Swensen and Mullin, 1997) and (iii) through the

ge-netic alteration of selected endophytic bacteria, or

direct incorporation of nitrogen-fixing genes (Dixon

et al., 1997; Gough et al., 1997). The reader is referred

to reviews in Ladha et al. (1997).

Seed treatments. Judging by past experience,

ap-plying bacterial seed treatments prior to planting does

not guarantee the establishment of a beneficial

endo-or exendo-orhizal flendo-ora (Frommel et al., 1993) nendo-or does it

always enhance yield (Volkmar and Bremer, 1998).

Introductions of non-local microfloras must compete

with established microbial communities in the soil,

the rhizosphere and within the plant. Both true seeds

and plants which are propagated vegetatively are

likely to carry enduring consortia of adapted

endo-phytes, a portion of which will be transferred to the

subsequent progeny. Niche specialization will ensure

that local communities are better positioned to

col-onize and retain niche dominance at the expense of

later introduced species. Our feeling, at the present

time, is that seed treatments are best suited to

aug-menting established consortia of microbial organisms

(fungal, bacterial and mycorrhizal) created as part of

a long-term strategy of harmonized crop (cultivar)

selection and management practices.

5. Conclusions

Current interest in beneficial rhizobacteria has

fo-cused on the exoroot and its associated rhizosphere

community. However, plants are also colonized by

a diverse array of endophytic bacteria which form

non-pathogenic relationships within plants. Positive

interactions between endophytes and their host plants

can result in a range of beneficial effects which are

similar if not complementary with those reported for

the exorhizobacteria. These include increased plant

growth and development, resistance to disease and

improvements in the host plant’s ability to withstand

environmental stresses (e.g. drought). Endophytes

offer the twin benefits of being acclimated to their

hosts, and present at seedling development and

rhi-zosphere initiation. These factors provide endophytes

with a competitive ecological advantage compared

to the resident ‘wild-type’ soil microflora that are

so often implicated in the failure of biological seed

treatments (biocontrol agents and growth promotion

amendments). However, much of the basic

informa-tion regarding endophyte community structure, their

principal functions, relative ecological stability, and

the organizing forces that govern their continuity, is

still lacking. If rhizobacteria are to be better utilized

in crop production systems, then one approach to

enable this to happen should involve the creation and

enhancement of sustainable, beneficial communities

of bacteria in the endo- as well as the exoroot.

References