Endophytic communities of rhizobacteria and the strategies
required to create yield enhancing associations with crops
A.V. Sturz
a,∗, J. Nowak
baPrince Edward Island Department of Agriculture and Forestry, PO Box 1600, Charlottetown, PEI, Canada C1A 7N3 bDepartment 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.
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.
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
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.
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.
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