Colonization of the Rhizosphere

APOPLASM

A. Colonization of the Rhizosphere

tions may be considered beneficial, neutral, or harmful to the plant, depending on the specific microorganisms and plants involved and on the prevailing environ- mental conditions.

Plant beneficial microbial interactions can be roughly divided into three categories. First, there are those microorganisms which, in association with the plant, are responsible for its nutrition (i.e., microorganisms that can increase the supply of mineral nutrients to the plant). This group includes dinitrogen-fixing bacteria such as those involved in the symbiotic relationships with leguminous plants (for example, Rhizobilrrr~ and Btudyrhizobiutn species) (see Chap. IO), with monocots (for example, Azo.spirill~ltn brasileme) or free-living nitrogen-fixing bacteria such as Klebsiella pneumoniae. In addition, there are a number of micro- bial interactions that increase the supply of phosphorous (for example, mycorrhi- zae) (see Chap. 9) and other mineral nutrients to the plant. Second, there is a group of microorganisms that stimulate plant growth indirectly. by preventing the growth or activity of plant pathogens. Such organisms are often referred to as hiocontrol agents, and they have been well documented. A third group of plant-beneficial interactions involve those organisms that are responsible for di- rect growth promotion-for example, by the production of phytohormones. Plant growth-promoting rhizobacteria (PGPR) or plant-beneficial microorganisms and their use to increase plant productivity have been the subject of several reviews (70-75), and examples are discussed below.

Neutral interactions are found extensively i n the rhizosphere of all crop plants. Saprophytic microorganisms are responsible for many vital soil processes, such as decomposition of organic residues in soil and associated soil nutrient mineralizationhrnover processes. While these organisms do not appear to bene- fit or harm the plant directly (hence the term neutml), their presence is obviously vital for soil nutrient dynamics and their absence would clearly influence plant health and productivity.

Detrimental interactions within the rhizosphere include the presence and action of plant pathogens and “deleterious rhizobacteria.”

Root exudates play a key role in determining host-specific interactions with, and the composition of, their associated rhizobacterial populations. Root exudates can attract beneficial organisms such as mycorrhizal fungi and PGPR (13,76), but they can also be equally attractive to pathogenic populations (77,78). As mentioned above, it is the balance between beneficial and detrimental microor- ganisms that ultimately governs plant nutrition and plant health. Before dis-

cussing some of the specific interactions mentioned above, consideration is given to the microbial colonization of the rhizosphere.

to support the theory of coevolution of plants and their associated rhizosphere microbial populations.

Root colonization can be considered to involve four stages. The initial stage of root colonization is the movement of microbes to the plant root surface. Bacte- rial movement can be passive, via soil water fluxes, or active, via specific induc- tion of flagellar activity by plant-released compounds (chemotaxis). The second step in colonization is adsorption to the root. This is a step required before anchor- ing and can be defined as nonspecific and based on electrostatic forces, whereas anchoring can be defined as the firm attachment of a bacterial cell to the root surface. Following adsorption and anchoring, specific and/or complex interac- tions between the bacterium and the host plant may ensue, which lead to induction of bacterial gene expression. Table 1 cites examples of root exudate components involved in these processes.

B. Specific Microbial Interactions

1. Beneficial Interactions

C I . Dinitroger1 Fixation. Species of Rhizobiutn and B~-n&rl~izobiunz have

long been known to induce the formation of root nodules on leguminous plants (see Chaps. 7 and 10). Once formed, a differentiated form of the bacterium, the bacteroid, converts atmospheric nitrogen into ammonia, which is then used as a nitrogen source by the plant. In return, the plant provides a carbon source to the bacteroid, probably in the form of dicarboxylic acid (74). Nodulation is host- specific, and each (Bmdy)r.hizobiunz species can nodulate only a restricted num- ber of legume species. Both nodulation and nitrogen fixation are complex pro- cesses, involving interactions between a number of bacterial genes and their gene products and plant products. Specific compounds released by roots of young le- gumes are involved in attracting these symbiotic bacteria to their roots (see Table 1). Following infection of the host root system, flavonoid compounds released by the root hair zone of the plant are believed to be responsible for the induction of bacterial nodulation (nod) genes in Rhizobium species (91), which results in the biosynthesis of Nod factors. Nod factors are a group of biologically active oligosaccharide signals whose effects on the host legume are similar to the early developmental symptoms of the Rhizobium-legume symbiosis, includ- ing root hair deformation and nodule initiation (92). The role of organic signaling molecules between plants and microorganisms and the biochemistry of the associ- ation between rhizobia and their host plants are discussed more thoroughly in Chaps. 7 and IO, respectively.

In addition to N,-fixing symbioses with legumes, associations between N2- fixing microorganisms and the roots of monocots such as cereals and forage grasses have been reported (93). However, the benefit of nitrogen fixation to nonlegumes was thought to be small, with studies showing that the contribution

Process Exudate component Microbial species Reference I . Movement of microbes to root surface

A. Passive

B. Active-induction of AageIlar activity (chemotaxis)

2. Adsorption 3. Anchoring

A. Bacterial appendages (pili. fimbriae) 8. Agglutination

C. Formation of cell aggregates 4. Gene expression

A. Nodulation genes-production of nod factors B. Virulence penes-production of virulence factors

Luteolin/Phenolics Acetosyringone Benzoate/arornatics Amino acids. nucleotides Serine

Unidentified Unidentified Unidentified sugars

and sugars

Lec t i n s

Flavonoids

Rfii,-cibiurn rnelilriti Agrohucterium tionefucieiis Arospirillimr spp.

Agrobucreriurn tumefuciriis Pseudomorius laclz~mans Pseuilomoncis aerugiriosa Pseudomnncis jhorescens Pseudonionas putida A;ospirilhcm briisilenw

Pseudomonos f7iioresrriis Pseudoinonas pufida Enterobiicter ciggloriieraris Rhirobiurn spp.

Brudl\rliirobium spp.

Agrobacteriirm fuinefuciens

80 81 82 83 83 83 83 83 84.85

86 8 7 3 8

89 YO,91.92

90 Table lists examples of root exudate components involved in chemotaxis. anchoring, and induction of gene expression.

of N, fixation by Azospirillurn to plant growth was minimal, with yield increases ranging from 5-18% (85). It was also found that mutants unable to fix nitrogen ( N t f ) were capable of increasing plant growth, like a wild-type N 2 fixer (94). It was suggested that the observed beneficial effects (increased yield, increased wa- ter and mineral uptake) of Azospirillum may derive from improvements to root development as a result of the action of phytohormones such as IAA, gibberellins, and cytokinin-like substances produced by the strain rather than from its nitrogen- fixing abilities (85,94,95). However, more recent work has initiated a reappraisal of the theory of N, fixation as a mechanism of plant growth promotion by Azospir- illurn spp. ( 8 5 ) . Most importantly perhaps, results indicate that graminaceous

plants are potentially capable of establishing associations with diazotrophic bacte- ria in which high ammonium-secreting Awspirillurn mutants provide the host with a source of nitrogen.

0. Mycorrhizrre. The biochemistry of the association between mycorrhi- zae and the plant is extensively discussed in Chap. 9. Arbuscular mycorrhizal (AM) fungi interact symbiotically with approximately 80% of all plant species (96). Mycorrhizal symbioses are present in most natural and agricultural ecosys- tems, where they are involved in many key processes-including nutrient cy- cling, maintenance of soil structure. plant health, and enhancement of nitrogen fixation by rhizobia (97). Their primary effect on the plant is the improvement of plant growth by increasing the supply of mineral nutrients from the soil to roots. Arbuscular mycorrhizal fungi are known to influence phosphorus (P) up- take and plant growth in P-deficient soils (98,99). Several mechanisms have been proposed to account for the increases in uptake of phosphorus, one of the most important being the acidification of the rhizosphere (100, I O l ) , which may explain how vesicular arbuscular mycorrhizal fungi increase the uptake of P as a result of plant utilization of ammonium (NHJ' -N). Higher P uptake as a result of plant utilization of NHJi occurs in both neutral and alkaline soils and is discussed further on. More efficient utilization of ammonium by mycorrhizal than nonmy- corrhizal plants has been shown by several authors ( 102,103) and may lead to increased H' secretion i n to the rhizosphere as a result (104,105). Rhizosphere pH has been reported to be altered by the form of nitrogen added both in thc presence and absence of mycorrhizae (102,106,107). It has also been reported that infection by ectotnycorrhizae significantly enhanced the capacity of plant roots to release H+ into a medium, which can increase the bioavailability o f com- pounds not readily soluble at higher pH (108). If a mycorrhizal plant induces a decrease in its rhizosphere pH, this effect may contribute to more P uptake by solubilizing calcium P and iron and aluminium phosphates, and thus increasing P availability to both the root and hyphae. I n general, the addition of nitrogen stimulates the uptake of P by the plant, especially when NH,+ is applied ( 109,l I O ) . Ortas et al. ( 1 1 I ) found that application of ammonium led to increased

plant dry weight and P content of sorghum as compared to plants treated with nitrate. These differences were enhanced by inoculation with mycorrhizae. It has also been shown that arbuscular mycorrhizal fungi can take up significant quanti- ties of Cu, Zn, and Cd ( I 12), providing a mechanism by which plants avoid exposure to toxic quantities of these metals. The improved phosphate, nitrogen, or micronutrient uptake by plants as a result of mycorrhizal associations also has secondary effects on the uptake of other ions, such as potassium, sulfate, and nitrate. In addition, the increased uptake of phosphate indirectly stimulates nodu- lation and nitrogen fixation (74), since rhizobia require substantial amounts of this element for their activity. The biochemistry of the association between my- corrhizae and plants is covered i n depth in Chap. 9.

c. Biocontrol. Evidence suggests that monoculture of crops eventually leads to decreased yields as a result of an increase i n plant pathogen numbers in the soil (74). Crop rotation has previously been employed as a method to alleviate this problem, by denying pathogens a suitable host for a period of time so that their numbers in soil decline. However, i t has also been noted that in some soil systems repeated monoculture eventually leads to a decrease in plant disease (74).

In these cases the disease-conducive soils are converted to disease-suppressive soils. This phenomenon has led to the isolation of a wide range of microbial biocontrol agents from disease-suppressive soils ( 1 13). Mechanisms by which these organisms are known to antagonize plant pathogens are varied, and some of these are discussed below. In some instances, control of a pathogen may result from a combination of two or more of these mechanisms.

I . PRODUCTION OF ANTIBIorICS. The production of secondary metabo-

lites with antimicrobial properties has long been recognized as an important factor in disease suppression (see Chap. 7). Metabolites with biocontrol properties have been isolated from a large number of rhizosphere microorganisms, including the fluorescent pseudomonads (Table 2). Further discussion is not given here since this is the subject of recent reviews (122,123).

11. PRODUCTION OF SIDEROPHORES. Many plant growth-promoting bac- teria, especially pseudomonads, produce high-affinity Fe3'-binding siderophores under conditions of low iron concentration ( 1 24,125). This may result in severe iron limitation in the rhizosphere, which could limit the growth of other rhizo- sphere bacteria and fungi. Many authors have demonstrated the importance of

siderophores in the inhibition of both fungal and bacterial pathogens (126-129).

This topic is discussed in Chap. 8.

111. PRODUCTION OF VOLATILE COMPOUNDS. Volatile compounds such as ammonia and hydrogen cyanide are produced by a number of rhizobacteria and are also believed to play a role in biocontrol. For example, Pseudomorfns jluorescens strain CHAO can produce levels of HCN that in vitro are toxic to

of Root Exudates 109

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pathogenic fungi such as T l ~ i e l u v i o p s i s h a s i c o h , and it is possible that this is the mechanism responsible for prevention of black root rot of tobacco in the field ( 1 30).

I V . PARASITISM. Lysis by hydrolytic enzymes excreted by microorgan- isms is a well-known feature of mycoparasitism. Chitinase and p-1,3 glucanase (laminarase) are particularly important enzymes secreted by fungal mycoparasites capable of degrading the fungal cell wall components, chitin, and

p-

I ,3 glucan

Many rhizobacteria are classified as chitinolytic and, for example, Serruticl tncwsescrtzs, which excretes chitinase, was found to be an effective biocontrol agent against Sclerotium roljkii (135). Similarly, Aerornonus cclviue was found to reduce disease caused by Rhizoctonia solani, Fusarium o.vy.spor~c~n, and Sclrro- tiurn roljkii (136). There is also evidence to support the role of p-1,3 glucanase in biocontrol of soil-borne plant pathogens (137).

(131-134).

v. COMPETITION FOR NUTRIENTS. Elad and Chet ( 1 38), studying biocon- trol of Pythiurn damping-off by rhizobacteria, suggested that competition for available carbon and nitrogen sources may account for observed disease reduc- tion. They found competition for nutrients between germinating oospores of Pyth- i r m e ~ p h c ~ r ~ i d r ~ t ~ ~ c r t ~ r t ~ ~ and bacteria (which was unique to isolated biocontrol strains) significantly correlated with suppression of the disease. It appeared that bacteria were competing with germinating oospores for available C and N. and by eliminating these resources, the bacteria effectively reduced oospore germination.

V I . COMPETITION FOR ECOLOGICAL N I C H E . An alternative mechanism involved in biocontrol is that of niche exclusion. For PGPR to function success- fully in the field, they must inevitably be able to establish themselves effectively on plant roots. Pseudornorlcls strains, for example, are able to establish on roots from inoculated seeds relatively easily. This is a major factor contributing to the success of these strains as biological control agents. However, root colonization is not the only criterion for defining a successful PGPR strain. Most diffusible root exudates are associated with the zone of root elongation, inducing coloniza- tion by PGPR in this zone. However, some studies have indicated establishment of populations along a greater. more undefined area of the root surface, including root tips, zones of elongation, and zones of lateral root emergence.

Since phytopathogenic bacteria occupy particular niches in the rhizosphere, it has been proposed that the deliberate application of a nonpathogenic mutant of the same species may prevent pathogen establishment through niche exclusion by the nonpathogenic mutant. An example of this was the deliberate release of a nonpathogenic Ice-. mutant of P.srudot~1onu.s syringue to compete with plant pathogenic lce' P. syringae to prevent frost damage (139,140). The Ice' (patho- genic) strain of this organism causes frost damage to plants by making an ice

nucleation protein that initiates ice crystallization at temperatures not normally favorable for ice formation. It was envisaged that deliberate release of the Ice- mutant of P . syrirrgrre from which the ice nucleation protein gene had been deleted could prevent frost damage by colonizing niches previously occupied by the Ice' strain. In the field, it was found that frost damage was indeed decreased following the application of the genetically modified Icc- strain.

v11. I N D U C E D DISEASE RESISTANCE. Another mechanism responsible for the biological control of plant disease is induced systemic resistance (ISR) or induced disease resistance. ISR protects the plant systemically following induc- tion with an inducing agent to a single part of the plant. The action of ISR is based on plant defense mechanisms that are activated by inducing agents (141).

ISR, once expressed, activates multiple potential defense mechanisms that in- clude increases in activity of chitinases,

p-

1,3-glucanases, peroxidases, and other pathogensis-related proteins (142); accumulation of antimicrobial low-molecular- weight substances such as phytoalexins (143), and the formation of protective biopolymers such as lignin, cellulose, and hydroxyproline-rich glycoproteins

(144-146). A single inducing agent can control a wide spectrum of pathogens.

I n cucumber, for example, treatment of the first leaf with a necrosis-forming organism protects the plant against at least 13 pathogens, including fungi, bacte- ria, and viruses (147). Caruso and Kuc (148) showed systemic protection in cu- cumber and watermelon resulting from induction with Colleototrichrrm orhicu- lore against challenge inoculum of the same pathogen. Many other cases have been reported, but widespread application has not been accomplished, as classical ISR employs pathogenic organisms as inducing agents. More recent work, how- ever, has demonstrated that some PGPR may act as inducing agents, leading to systemic protection against pathogens. In response to earlier observations of PGPR-mediated ISR in the greenhouse, Wei et al. (149) carried out three 2-year field trials to determine the capacity of PGPR to induce systemic resistance against cucumber diseases. Results indicated that PGPR-mediated ISR was opera- tive under field conditions, with consistent effects against challenge-inoculated angular leaf spot and naturally occurring anthracnose. Furthermore, ISR induced early-season plant growth promotion and increased yield.

In these studies, the pathogen and the resistance-inducing PGPR were ap- plied at separate locations on the plant, excluding direct antibiosis and competi- tion as mechanisms of disease suppression (149).

d . Prodrction o f P h t Growth-Promoting Compounds. A large num- ber of rhizobacteria, such as strains of A z o s p i r i l h m . Azotohocter, P.~eurlor,lorlrl,s and Bucillus, have been shown to produce plant growth-promoting compounds such as indoleacetic acid (IAA), gibberellin, and cytokinin-like substances (93,95,150- 152). The presence of such compounds in the rhizosphere appears to stimulate plant growth directly.

2. Detrimental Microbial Interactions

cl. P l m f Parhogens. Interactions between microbial pathogens and

plants are in general host-specific and consequently have been considered to be influenced by root exudate components. There are examples where amino acids, sugars, and other exudate components have been shown to directly stimulate pathogens (77,153). Stimulation of chlamydospore germination of Fuscrriurn so- lan i f i pha s e o l i (154) and Thielviopsis hnsicola (155) was found to occur at the surface of bean seeds and roots but not in soil distant from the host. Phyrophora, PJIrhium, A p h a n o m y c e s , and other examples of pathogenic oomycetes have motile zoospores and consequently tend to accumulate around roots via chemotactic or electrostatic responses to root exudates (156). Germ tubes from spores of Phyto- phora cir~nnrnorni have also been shown to exhibit chemotrophisnl, becoming oriented toward the region of elongation of susceptible roots. Exudate-induced interactions unfavorable to propagule germination, growth, and colonization of roots by plant pathogens are often associated with the activity of other miroorgan- isms in the rhizosphere. Since all these organisms occupy the same microhabitat, there is inevitably direct competition for nutrients and ecological niche. For ex- ample, Paulitz ( 1 57) found that hyphal growth from soil-produced sporangia of PyrhiLtnl ulrirnurn was stimulated by volatiles from germinating pea seeds, and this stimulation was reduced when seeds were treated with PseLc~lomonas ,puo- resc'ens N 1 R. It was thus suggested that N 1 R may reduce damping-off by compet- ing for and using volatile exudates from germinating pea seeds.

In addition, plant pathogens may be antagonized by compounds released by plant roots, plant residues, and other microorganisms present in the rhizosphere.

Further, the survival and activity of specific plant pathogens may be affected by the action of antagonists present in the rhizosphere. To reiterate, it is the balance between plant pathogenic and antagonistic microorganisms that determines the effect on plant health.

h. Deleterious Rhizohacreria. Rhizobacteria that inhibit plant growth

without causing disease symptoms are frequently referred to as deleterious rhizo- bacteria (DRB), or minor pathogens. DRB can inhibit shoot or root growth with- out causing any other visual symptoms (158-160) and may be partly responsible for growth and yield reductions associated with continuous monoculture

(160,161). DRB implicated in yield decline in a number of crops have been re- viewed by Nehl et al. (35). Several mechanisms for growth inhibition by DRB have been proposed, the most likely being the production of phytotoxins such as cyanide (162,163) and other volatile and nonvolatile compounds as yet uniden- tified (164,165). An alternative mechanism by which DRB inhibit plant growth may be through the production of phytohormones (160). IAA produced by DRB has been shown to inhibit root growth in sugarbeet (166) and blackcurrant (167).

DRB may also compete with the plant and beneficial rhizobacteria for nutrients,