Exoroot

Figure 1. Parallel action of disease suppression mechanisms operating within the host plant (endoroot) and in the surrounding soil (exoroot).

reasonably comfortably within the ambit of allelopathy (see Keel and Defago, 1997;

Five common mechanisms are usually cited, namely:

i) Resource(niche) competition (or exploitation) (O'Sullivan and O'Gara, 1992; Stephens et al., 1993; Whipps, 2001) - for example, siderophore (chelator)-producing bacteria with high affinities for, and capable of sequestering, specific mineral elements, can inhibit phytopathogens with the same requirement if that mineral is limited in the soil (Schroth and Hancock, 1982; Dowling et al, 1996; Loper and Henkels, 1997, 1999).

ii) Antibiosis action - through the production of specific or non-specific microbial metabolites with antibacterial, antifungal and anti-nematode activity (Levy and Carmelli, 1995; Fujimoto et al., 1995; Raaijmakers et al., 2002; Thomashow et al., 1997). To-date, several antibiotic substances have been identified of which those produced by the pseudomonads have been particularly well characterized. of these, antibiotics identified with biocontrol properties include the phloroglucinols, phenazine derivatives, pyoluteorin, pyrrolnitrin, cyclic lipopeptides and hydrogen cyanide (Haas and Keel, 2003). Among the other antibiotics characterized are agrocin 84 (Agrobacterium sp.), herbicolin A (Erwinia sp.), iturin A, surfactin, and zwittermicin A (Bacillus sp.) and xanthobacin (Stenotrophomonas sp.) (Hashidoko et al., 1999; He et al., 1994; Sayre and Starr, 1988; Thomashow et al., 1997; Silo-Suh et al., 1994). A comprehensive account of bacterially produced antibiotics may be found in Raaijmakers et al. (2002).

iii) Lytic enzyme action - a feature of several bacteria with proven biocontrol ability, and generally involves the direct degradation of pathogen cell wall material, or the disruption of a particular developmental stage. Thus, for example, chitinase production by Serratia plymuthica has been reported to inhibit spore germination and germ-tube elongation in Botrytis cinerea (Frankowski et al., 2001), while 6-1,3-glucanase synthesized by Paenibacillus sp. and Streptomyces sp. can lyse fungal cell walls of Fusarium oxysporum f. sp. cucumerinum (Singh et al., 1999). Other enzymes produced by bacteria with biocontrol activity include hydrolase (Chernin and Chet, 2002), laminarinase (Lim et al., 1991) and protease (Kamensky et al., 2003).

iv) Induced systemic resistance (ISR) in plants (Wei et al., 1991; Tuzun and Kloepper, 1994) - whereby non-pathogenic rhizobacterial stimulation of defence-related genes is elicited through the encoded production of jasmonate (van Wees et al., 1999), peroxidase (Jetiyanon et al., 1997) or enzymes involved in the synthesis of phytoalexins (van Peer et al., 1991). Though no specific ISR-eliciting signal has been identified, thus far, evidence for the involvement of lipopolysaccharides, siderophores and phloroglucinols has been submitted (Hoffland, et al., 1995; Leeman et al., 1995, 1996; Maurhofer et al., 1994; van Wees et al., 1997), and, v) Root camouflage (Gilbert et al., 1994) - proposed as a mechanism to explain the observation that certain rhizobacterial populations in disease resistant cultivars are able to minimize the 'attractive' nature of the host's root system so masking its presence to potential plant pathogens by restricting local population density development. Such microbial systems may operate in tandem with those that desensitize the chemoperception systems of microorganisms in the root zone, through the over production of chemical stimulii (Armitage, 1992; Dusenbery, 1992).

The parallel operation of all these biocontrol mechanisms in a four dimensional soil space makes their action and interaction difficult to follow. Biocontrol strains only occupy a small fraction of the root surface, in microcolonies spread out unevenly along the root surface (Bowen and Rovira, 1976; Normander et al., 1999). Disease suppression, when it occurs through antibiosis, is most likely restricted to local action only, and most probably at sub-inhibitory levels. Even so, antibiotics can cause intense physiological effects upon neighbouring organisms at subinhibitory concentrations. Quinolone and macrolide antibiotics have been reported to block cell- to cell signaling, and the production of virulence factors in P. aeruginosa (Grimwood et al., 1989; Tateda et al., 2001). Similarly, subinhibitory concentrations of antibiotics can suppress adherence mechanisms in bacteria (Breines and Burnham, 1994), and the production of extracellular virulence factors in bacteria (Herbert et al., 2001). Accordingly, secondary metabolites can impact soil microbial ecosystems in a variety of ways, and at a variety of levels (Haas and Keel, 2003).

3.3. Partitioning of disease suppressive bacteria in the endo and exoroot

Most research into soil bacterial communities has been restricted to the exoroot - that fraction of the microfloral community found in the rhizoplane, plant rhizosphere or root zone soil (Haas and Keel, 2003). Endoroot bacteria have largely been ignored, despite plant-bacteria interactions extending into the endoroot of all plants (Conn et al., 1997; Bensalim et al., 1998). The frequent recovery of communities of endophytic bacteria, in the absence of any pathological condition (Chanway, 1996) and the finding that bacterial endoplant communities are capable of mediating against phytopathogen invasion (Benhamou et al., 1996) has led to the suggestion that plants may have co-opted bacteria as part of a disease suppressive response to phytopathogen attack.

Several instances have been reported of endophytic bacteria as effective biocontrol agents (van Buren et al., 1993; Brooks et al., 1994). van Peer et al. (1990) found that endo- and exoroot bacteria from the same genera formed discrete sub-populations, each suited to colonizing their respective environmental niches. Tissue-specific relationships can form between communities of bacterial endophytes and their host plant, and endobacteria have been shown to adapt functionally to certain tissue sites and among certain tissue types (Sturz et al., 1999). Unfortunately, the population densities of endophytic bacteria tend to be highly variable among plant tissues and so may be of little practical value in terms of affording plants a comprehensive first line of defence to pathogen attack.

4. 'ENGINEERING' BENEFICIAL MICROBIAL ALLELOPATHIES

4.1. Anthropogenic intrusions

The premise underlying most anthropogenic biocontrol systems is the notion that it is possible to encourage the occurrence and development of beneficial rhizobacterial allelopathies in the root zone, primarily through the direct application of specific biocontrol agents, and or soil conditioning amendments (Sturz and Christie, 2003). It must be acknowledged at the outset that such systems have had varying degrees of success.

4.2. Inundative approaches in biological control

In general, anthropogenic attempts to import biological control agents into the field have been through the inundative application of super quantities of a few key biocontrol or plant growth promoting agents. These have been applied directly as drenches or sprays, or alternatively as a cell suspensions incorporated in mulches or compost material. Carriers such as granular peat formulations, mineral soils (Chao and Alexander, 1984), bacterial encapsulations within polymer gels (Bashan, 1986) or in natural gum or talc mixtures (Kloepper and Schroth, 1981) have also been tried.

Perhaps, in hindsight, it is not surprising that such inundative approaches have been relatively unsuccessful, with most biocontrol agents failing to fulfill their initial promise. Such failures have usually been attributed to poor competence of biocontrol agents in the infection court and the difficulties associated with the instability of biocontrol agents in culture (Schroth et al., 1984; Weller, 1988); not least because in the case of bacterial agents, the accumulation of extracellular signalling molecules within large population densities of individual strains, can modulate a diverse range of metabolic processes, some of which are incompatible with the goals of phytoprotection (see above).

However, the use of single antagonists may itself be an inappropriate strategy, arising from the belief that a given plant disease can be attributed to a single pathogen only (Baker, 1987). Such one-on-one syndrome concepts follow from the belief that successful control of a pathogen is achievable with a single fungicide, or by single-factor resistance, coupled with the observation that single antagonists have often provided effective control in presumptive tests for antagonists in in vitro studies, or as biocontrol agents applied to sterilized soil (Baker, 1987)

Needless to say, the very practice of applying massive quantities of a single bacterial species to the infection court will not only alter the putative biocontrol agent's physiology, but also its niche behaviour. Perhaps, more intriguingly, it may also garner a general and antagonistic response from the resident population.

Several attempts at engineering bacterial strains with more reliable biocontrol performance have been tried, whereby biosynthetic genes for various antibiotics have been designed to be constitutively over-expressed. On occasion, such engineered strains have provided improved plant protection in the soil microcosm (Delany et al., 2001; Ligon, et al., 2000; Timms-Wilson et al., 2000). However, in long term field evaluations, engineered derivatives have also lacked consistency; loss of stable performance and lack of superiority to wild-type strains being cited as the principal reasons for failure (Bakker et al., 2002).

Although it would be premature to generalize the findings of such studies, it appears that the engineering of a single trait (antibiotic production) in a single biocontrol strain can not overcome the problem of inconsistent performance in the field, given the multi-factor nature of biocontrol mechanisms and the potential for interaction with wild-type species in the soil microbial community.

While one-on-one antagonism may indeed be the sole operating mechanism involved in microbial disease suppression, an equally valid interpretation might be that the inundative addition of biocontrol agents can stimulate a general antagonistic response from the autochthonous microbial population to the 'invader' (inundating) species. In this circumstance and irrespective of any inconsistencies in the field performance of biocontrol agents attributed to unfavourable edaphic factors - such as temperature, soil moisture, pH, clay content, soil type - biocontrol success or failure may simply be due to the resident community's antagonistic response following the inundative insertion of a non-indigenous species. Consequently, both pathogen and biocontrol agent are inhibited in collateral fashion and to various degrees; a scenario that is congruent with the defensive mutualism theory proposed by Clay (1988).

4.3. Modifying Soil Agro-ecosystems

The extent to which producers can develop beneficial root zone allelopathies amongst microbial communities will depend largely upon the resilience of the soil in question (Szabolcs, 1994) and the type of crop management and tillage systems being practised (Sturz and Christie, 2003). Plant species are known to apply a selective and specific influence on microbial diversity in the rhizosphere through their differential root exudate spectra (Grayston et al., 1998), and the plastic nature of the relationship between resident microbial communities. Thus the level of disease suppressiveness in a soil is eminently amenable to deformation through the use of selected cultural practices. This regardless of the inherent capacity of 'natural' soil microbial ecosystems to buffer anthropogenic interference.

Crop management systems are regularly used to distort agro-ecosystems through, for example, the use of tillage operations, alternate cropping systems, monoculture, crop rotation length, fertilizer and organic amendments, and various crop protection chemistries. The management of soil microbial communities for crop yield maximization appears to involve, in part, the creation of short term chaos in the microbial community through the application of a plethora of perturbation stresses (Odum et al., 1979). Moderate levels of 'input perturbation' are considered to improve ecosystem performance, while higher levels of perturbation stress result in performance loss.

input perturbations have commonly been used to modify soil microbial agro-ecosystems at the expense of pathogen populations. The subsequent variation in habitat and increase in niche heterogeneity - though on a microscale and at multiple sites along the root - is believed to encourage microbial biodiversity and consequently increase the potential for root zone competition (Smucker, 1993; Andrews and Harris, 2000). Thus, for example, increasing soil acidity (Davis and Callihan, 1974, Sturz et al., 2003), applying irrigation soon after tuber initiation (Lapwood et al., 1973; Oestergaard and Nielsen, 1979) and the addition of soil amendments, green manures and mulches (Tremblay and Beauchamp, 1998) have all been relatively successful in reducing the development common scab on potatoes.

Disease suppression has also been achieved against a wide range of pathogens by incorporating green manures (plough-down crops) (Tu and Findlay, 1986), animal manures (Gorodecki and Hadar, 1990) and composts (including organic solid wastes) (Nelson and Hoitink, 1983; Cohen et al., 1998) into field soils. All these amendments can encourage aggressive competition among microbial communities (Hoitink and Boehm, 1999; Hoitink and Fahy, 1986; Hoitink et al., 1997), with the added effect that manure and compost decomposition can release both volatile and non-volatile toxic compounds that inhibit phytopathogenic nematodes (Sayre et al., 1965; Abawi and Widmer, 2000) and reduce the survival rates of pathogenic microbes (De Brito et al., 1995; Chen et al., 1987 a, b).

Though time consuming, unfashionable and often slow to show effect, traditional crop production practices that involve environmentally sustainable practices, such as conservation tillage (Sturz et al., 1997; Bockhus and Shroyer, 1998), 'creative' fallowing options (Sturz et al., 2001), manuring (Hoitink and Boehm, 1999), long term crop rotations (Peters et al., 2003) and compatible cropping systems (Sturz et al., 2003), can yield plant health and crop yield benefits. Whether such knowledge-based, time-intensive management practices can be made more popular remains the challenge.

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