Journal of Experimental Botany, Vol. 52, No. 90001, pp. 487-511,
March 2001
© 2001 Oxford University Press
Microbial interactions and biocontrol in the rhizosphere
Plant Pathology and Microbiology Department, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK
Received 23 March 2000; Accepted 13 July 2000
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Abstract |
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 Top Abstract IntroductionBacteria-bacterial pathogen...Bacteria-fungal pathogen...Fungal-protozoan interactionsFungal-bacterial pathogen...Fungal-fungal pathogen...Multiple microbial interactionsConclusions and future...References |
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The loss of organic material from the roots provides the energy for the development of active microbial populations in the rhizosphere around the root. Generally, saproptrophs or biotrophs such as mycorrhizal fungi grow in the rhizosphere in response to this carbon loss, but plant pathogens may also develop and infect a susceptible host, resulting in disease. This review examines the microbial interactions that can take place in the rhizosphere and that are involved in biological disease control. The interactions of bacteria used as biocontrol agents of bacterial and fungal plant pathogens, and fungi used as biocontrol agents of protozoan, bacterial and fungal plant pathogens are considered. Whenever possible, modes of action involved in each type of interaction are assessed with particular emphasis on antibiosis, competition, parasitism, and induced resistance. The significance of plant growth promotion and rhizosphere competence in biocontrol is also considered. Multiple microbial interactions involving bacteria and fungi in the rhizosphere are shown to provide enhanced biocontrol in many cases in comparison with biocontrol agents used singly. The extreme complexity of interactions that can occur in the rhizosphere is highlighted and some potential areas for future research in this area are discussed briefly.
Key words: Bacteria, biocontrol, fungi, roots, soil.
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Introduction |
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TopAbstractIntroductionBacteria-bacterial pathogen...Bacteria-fungal pathogen...Fungal-protozoan interactionsFungal-bacterial pathogen...Fungal-fungal pathogen...Multiple microbial interactionsConclusions and future...References |
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As seeds germinate and roots grow through the soil the loss of organic material provides the driving force for the development of active microbial populations around the root, known as the rhizosphere effect (Whipps, 1990
; Morgan and Whipps, 2001). Although the stimulation in microbial activity is a general phenomenon largely involving saprotrophs, specific groups of symbionts may be selectively enhanced. For example, mutualistic biotrophic symbioses may develop between Rhizobia and legumes, and mycorrhizal fungi may interact with their plant hosts. However, antagonistic symbioses between pathogens and roots can also form resulting in disease. The microbial interactions taking place in the spermosphere and rhizosphere associated with disease development and especially biocontrol of these diseases form the background of this review.
Interest in biological control has increased recently fuelled by public concerns over the use of chemicals in the environment in general, and the need to find alternatives to the use of chemicals for disease control. The key to achieving successful, reproducible biological control is the gradual appreciation that knowledge of the ecological interactions taking place in soil and root environments is required to predict the conditions under which biocontrol can be achieved (Deacon, 1994; Whipps, 1997a) and, indeed, may be part of the reason why more biocontrol agents are reaching the market-place (Fravel, 1999; Whipps and Lumsden, 2001; Whipps and Davies, 2000). This type of work requires a study not only of any potential biocontrol agent per se but also its interactions with the crop, the natural resident microbiota and the environment as well. In this regard, it is well known that some soils are naturally suppressive to some soil-borne plant pathogens such as Fusarium oxysporum Schlect.: Fr. Emend. Snyder & Hansen, Gaeumannomyces graminis (Sacc.) v. Arx & Oliver, Pythium and Phytophthora species and this suppression relates to both physicochemical and microbiological features of the soil (Whipps, 1997a). Importantly, a soil that is suppressive to one pathogen is not necessarily suppressive to another, and so specificity in the soil–plant–microbe interactions for disease suppression exists. Modern methods for analysing microbial community structures may prove particularly valuable to help define the key organisms or groups of organisms responsible for such natural suppression as well as for monitoring the spread and impact of introduction of specific biocontrol agents or other management practices on natural microbial populations (Duineveld et al., 1998; Natsch et al., 1998; Abassi et al., 1999; Buyer et al., 1999; Gamo and Shoji, 1999; Mazzola, 1999; Shiomi et al., 1999; Tiedje et al., 1999; Smit et al., 1999; Postma et al., 2000). Significantly, disease suppression can also be achieved by manipulation of the physicochemical and microbiological environment through management practices such as the use of soil amendments, crop rotations, use of fumigants or soil solarization. However, at present, greatest interest resides with the development and application of specific biocontrol agents for the control of diseases on seeds and roots and the interaction of these with pathogens and hosts, and will form the focus of this paper. There have been numerous reviews in recent years on this topic (see Whipps, 1997a, b, c; Punja, 1997; van Loon, 1997; Burges, 1998; Boland and Kuykendall, 1998; Harman and Kubicek, 1998; Funck Jensen and Lumsden, 1999; Hoitink and Boehm, 1999; Mathre et al., 1999, and references therein) and so only selected recent examples, illustrating key features of biocontrol on seeds and roots, particularly the different modes of action, will be discussed whenever possible. Modes of action include: inhibition of the pathogen by antimicrobial compounds (antibiosis); competition for iron through production of siderophores; competition for colonization sites and nutrients supplied by seeds and roots; induction of plant resistance mechanisms; inactivation of pathogen germination factors present in seed or root exudates; degradation of pathogenicity factors of the pathogen such as toxins; parasitism that may involve production of extracellular cell wall-degrading enzymes, for example, chitinase and ß-1,3 glucanase that can lyse pathogen cell walls (Keel and Défago, 1997; Whipps, 1997a). None of the mechanisms are necessarily mutually exclusive and frequently several modes of action are exhibited by a single biocontrol agent. Indeed, for some biocontrol agents, different mechanisms or combinations of mechanisms may be involved in the suppression of different plant diseases.
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Bacteria–bacterial pathogen interactions |
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TopAbstractIntroductionBacteria-bacterial pathogen...Bacteria-fungal pathogen...Fungal-protozoan interactionsFungal-bacterial pathogen...Fungal-fungal pathogen...Multiple microbial interactionsConclusions and future...References |
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In the last few years there have been relatively few studies of bacteria applied to seeds and roots for the purpose of controlling bacterial diseases. One example, is the application of non-pathogenic strains of Streptomyces to control scab of potato (Solanum tuberosum L.) caused by Streptomyces scabies (Thaxter) Waksman and Henrici (Ryan and Kinkel, 1997
; Neeno-Eckwall and Schottel, 1999). Here biocontrol may operate through antibiosis or competition for space or nutrients in the rhizosphere. In contrast, Pseudomonas fluorescens (Trevisan) Migula F113 was shown to control the soft rot potato pathogen Erwinia carotovora subsp. atroseptica (van Hall) Dye by production of the antibiotic 2,4-diacetylphloroglucinol (DAPG) and, through use of co-inoculation experiments with mutants lacking DAPG production, that competition was not a feature of biocontrol in this system (Cronin et al., 1997). Some evidence was also obtained that siderophore production by P. fluorescens F113 may play a role in biocontrol of potato soft rot under iron-limiting conditions, but DAPG appears to be the major biocontrol determinant. Pseudomonas species may also control crown gall disease in many dicotyledonous plants caused by Agrobacterium tumefaciens (Smith & Townsend) Conn (Khmel et al., 1998). However, the classic, and still commercially successful, bacterial-based biocontrol system is the use of non-pathogenic Agrobacterium strains to control Agrobacterium tumefaciens. Long-term molecular and ecological studies of this control system have identified how the biocontrol works and have also allowed potential problems associated with its use in the field to be overcome. The most widely used non-pathogenic Agrobacterium strain K84 produces a highly specific antibiotic agrocin 84, which is encoded by plasmid pAgK84. Inundative inoculation of Agrobacterium strain K84 to roots by dipping in cell suspensions prior to exposure to the pathogen effectively controls those strains of pathogen susceptible to agrocin 84. However, because there is a risk that plasmid pAgK84 could be transferred to pathogenic strains and reduce effectiveness of control (Vicedo et al., 1996; Stockwell et al., 1996; López-López et al., 1999), a transfer deletion mutant of K84, K1026 has been constructed (Jones et al., 1988). Strain K1026 is as efficient as K84 in biocontrol of both strains susceptible to agrocin 84 and those resistant to agrocin 84 (Jones and Kerr, 1989; Vicedo et al., 1993) and so, clearly, production of agrocin 84 is not the only mechanism of biocontrol. Production of other antibiotics such as agrocin 434 or ALS 84 may play a part (Peñalver et al., 1994; McClure et al., 1998), but the ability to survive and compete on roots may also be important. Studies where pathogenic cells were co-inoculated with K84 or K1026 resulted in survival of the pathogen on roots up to 8 months later, although no symptoms were present, providing evidence that the non-pathogenic strains prevented disease expression rather than killing pathogen cells directly (Peñalver and Lopez, 1999; Johnson and DiLeone, 1999). |
Bacteria–fungal pathogen interactions |
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TopAbstractIntroductionBacteria-bacterial pathogen...Bacteria-fungal pathogen...Fungal-protozoan interactionsFungal-bacterial pathogen...Fungal-fungal pathogen...Multiple microbial interactionsConclusions and future...References |
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The volume of literature in this area continues to increase at a rapid rate, stimulated by the increasing ease with which molecular techniques can be applied to answer questions concerning distribution, and occurrence and relative importance of specific modes of action. Some examples of the different types of bacteria-fungal pathogen interactions examined in the spermosphere and rhizosphere in just the last three years are given in Table 1
. Although a range of different bacterial genera and species have been studied, the overwhelming number of papers have involved the use of Pseudomonas species. Clearly, Pseudomonas species must have activity but it begs the question as to the features that make this genus so effective and the choice of so many workers. Pseudomonads are characteristically fast growing, easy to culture and manipulate genetically in the laboratory, and are able to utilize a range of easily metabolizable organic compounds, making them amenable to experimentation. But, in addition, they are common rhizosphere organisms and must be adapted to life in the rhizosphere to a large extent (deWeger et al., 1995; Marilley and Aragno, 1999). Having appropriate ecological rhizosphere competence may be a key feature for reproducible biological control activity in the spermosphere and rhizosphere. This criterion is already widely appreciated for many fungal biocontrol agents (see later). A few specific examples of the modes of action involved with bacterial biocontrol of fungal pathogens in the rhizosphere and spermosphere are given below.
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Antibiosis
There are numerous reports of the production of antifungal metabolites (excluding metal chelators and enzymes) produced by bacteria in vitro that may also have activity in vivo. These include ammonia, butyrolactones, 2,4-diacetylphloroglucinol (Ph1), HCN, kanosamine, Oligomycin A, Oomycin A, phenazine-1-carboxylic acid (PCA), pyoluterin (Plt), pyrrolnitrin (Pln), viscosinamide, xanthobaccin, and zwittermycin A as well as several other uncharacterized moieties (Milner et al., 1996
; Keel and Défago, 1997; Whipps, 1997a; Nielson et al., 1998; Kang et al., 1998; Kim et al., 1999; Thrane et al., 1999; Nakayama et al., 1999). To demonstrate a role for antibiotics in biocontrol, mutants lacking production of antibiotics or over-producing mutants have been used (Bonsall et al., 1997; Chin-A-Woeng et al., 1998; Nowak-Thompson et al., 1999). Alternatively, the use of reporter genes or probes to demonstrate production of antibiotics in the rhizosphere is becoming more commonplace (Kraus and Loper, 1995; Raaijmakers et al., 1997; Chin-A-Woeng et al., 1998). Indeed, isolation and characterization of genes or gene clusters responsible for antibiotic production has now been achieved (Kraus and Loper, 1995; Bangera and Thomashow, 1996; Hammer et al., 1997; Kang et al., 1998; Nowak-Thompson et al., 1999). Significantly, both Phl and PCA have been isolated from the rhizosphere of wheat following introduction of biocontrol strains of Pseudomonas (Thomashow et al., 1990; Bonsall et al., 1997; Raaijmakers et al., 1999), finally confirming that such antibiotics are produced in vivo. Further, Ph1 production in the rhizosphere of wheat was strongly related to the density of the bacterial population present and the ability to colonize roots (Raaijmakers et al., 1999). PCA from Pseudomonas aureofaciens Kluyver Tx-1 has even been used as a direct field treatment for the control of dollar spot (Sclerotinia homeocarpa F. T. Bennett) on creeping bentgrass (Agrostis palustris Hudson) (Powell et al., 2000).
Antibiotic production by bacteria, particularly pseudomonads, seems to be closely regulated by a two-component system involving an environmental sensor (presumably a membrane protein) and a cytoplasmic response factor (Keel and Défago, 1997). Mutation in either gene has similar multiple effects on antibiotic production. For example, P. fluorescens Pf-5 with a mutation in the apdA sensor gene lost the ability to produce HCN, Plt and Pln (Hrabak and Willis, 1992; Corbell and Loper, 1995) and P. fluorescens CHA0 with a defect in the gacA response gene lost the ability to produce Phl, Plt and HCN as well as protease and phospholipase C (Laville et al., 1992; Sacherer et al., 1994). However, the environmental signals that control the two-component system are unknown. Interestingly, the gacA gene is required for biocontrol activity in P. fluorescens CHA0 in the rhizosphere of dicotyledous plants, but not in those of the Gramineae (Schmidli-Sacherer et al., 1997), although the mechanisms are unclear.
Other two-component signalling phenomena may also be involved in PCA production by pseudomonads on roots. Pseudomonas aureofaciens 30-84 is a biocontrol agent of take-all disease of wheat (Triticium aestivum L.) caused by Gaeumannomyces graminis var. tritici Walker and operates through inhibiting growth of the pathogen by production of PCA (Pierson III and Pierson, 1996). In this system, pathogen growth on the root increases root exudation and this results in an increase in the population of P. aureofaciens 30-84 and other bacteria in the infection zone. Consequently, there is an increase in the level of the signal molecule N-acyl-L-homoserine lactone (HSL) produced at low levels constitutively by the phzI gene, in the rhizosphere which is sufficient to switch on the PCA synthesis pathway in P. aureofaciens 30-84 controlled by the phzR gene. The resulting PCA production inhibits further growth of the pathogen. This explains why P. aureofaciens 30-84 does not reduce the number of infection sites on the roots, but inhibits secondary growth of the pathogen. Significantly, HSL from other members of the rhizosphere microbial community can contribute to PCA production in P. aureofaciens 30-84 raising the question of the significance of interpopulation signalling on biocontrol and perhaps the enhanced performance of certain strains of bacteria when introduced with mixtures of other bacterial biocontrol strains (Pierson and Weller, 1994). In addition, antibiotic production in Pseudomonas spp. may be further controlled by the activity of housekeeping sigma factors encoded by rpoS or rpoD genes (Sarniguet et al., 1995; Schnider et al., 1995), illustrating the complexity of these regulatory systems.
Interestingly, signalling between pathogenic fungi and potential biocontrol bacteria has also been detected. In one case, trehalose derived from Pythium debaryanum Hesse up-regulated genes in its biocontrol strain Pseudomonas fluorescens ATCC 17400 (Gaballa et al., 1997) and yet in another example Pythium ultimum Trow caused a down-regulation of five gene clusters of P. fluorescens F113 which provides biocontrol of this pathogen in the rhizosphere of sugar beet (Beta vulgaris L.) (Fedi et al., 1997). These findings may be of considerable significance for bacterial–fungal interactions in general and has major implications for the control of gene expression in complex microbial communities.
Competition for iron
Although competition between bacteria and fungal plant pathogens for space or nutrients has been known to exist as a biocontrol mechanism for many years (see Whipps, 1997a, for references) the greatest interest recently has involved competition for iron. Under iron-limiting conditions, bacteria produce a range of iron chelating compounds or siderophores which have a very high affinity for ferric iron. These bacterial iron chelators are thought to sequester the limited supply of iron available in the rhizosphere making it unavailable to pathogenic fungi, thereby restricting their growth (O'Sullivan and O'Gara, 1992; Loper and Henkels, 1999). Recent studies have clearly shown that the iron nutrition of the plant influences the rhizosphere microbial community structure (Yang and Crowley, 2000). Iron competition in pseudomonads has been intensively studied and the role of the pyoverdine siderophore produced by many Pseudomonas species has been clearly demonstrated in the control of Pythium and Fusarium species, either by comparing the effects of purified pyoverdine with synthetic iron chelators or through the use of pyoverdine minus mutants (Loper and Buyer, 1991; Duijff et al., 1993). Pseudomonads also produce two other siderophores, pyochelin and its precursor salicylic acid, and pyochelin is thought to contribute to the protection of tomato plants from Pythium by Pseudomonas aeruginosa (Schroeter) Migula 7NSK2 (Buysens et al., 1996). However, siderophores are not always implicated in disease control by pseudomonads (Schmidli-Sacherer et al., 1997; Ongena et al., 1999). The dynamics of iron competition in the rhizosphere are often complex. For example, some siderophores can only be used by the bacteria that produce them (Ongena et al., 1999), whereas others can be used by many different bacteria (Loper and Henkels, 1999). Different environmental factors can also influence the quantity of siderophores produced (Duffy and Défago, 1999). There is also the further complication that pyoverdine and salicylate may act as elicitors for inducing systemic resistance against pathogens in some plants (Métraux et al., 1990; Leeman et al., 1996b).
Parasitism and production of extracellular enzymes
The ability of bacteria, especially actinomycetes, to parasitize and degrade spores of fungal plant pathogens is well established (El-Tarabily et al., 1997). Assuming that nutrients pass from the plant pathogen to bacteria, and that fungal growth is inhibited, the spectrum of parasitism could range from simple attachment of cells to hyphae, as with the Enterobacter cloacae (Jordan) Hormaeche & Edwards–Pythium ultimum interaction (Nelson et al., 1986) to complete lysis and degradation of hyphae as found with the Arthrobacter–Pythium debaryanum interaction (Mitchell and Hurwitz, 1965). If fungal cells are lysed and cell walls are degraded then it is generally assumed that cell wall-degrading enzymes produced by the bacteria are responsible, even though antibiotics may be produced at the same time. Considerable effort has gone into identifying cell wall-degrading enzymes produced by biocontrol strains of bacteria even though relatively little direct evidence for their presence and activity in the rhizosphere has been obtained. For example, biocontrol of Phytophthora cinnamomi Rands root rot of Banksia grandis Willd. was obtained using a cellulase-producing isolate of Micromonospora carbonacea Luedemann & Brodsky (El-Tarabily et al., 1996) and control of Phytophthora fragariae var. rubi Hickman causing raspberry root rot was suppressed by the application of actinomycete isolates that were selected for the production of ß-1,3-, ß-1,4- and ß-1,6-glucanases (Valois et al., 1996). Chitinolytic enzymes produced by both Bacillus cereus Frankland and Pantoea (Enterobacter) agglomerans (Beijerinck) Gavini et al. also appear to be involved in biocontrol of Rhizoctonia solani Kühn (Chernin et al., 1995, 1997; Pleban et al., 1997). Tn5 mutants of E. agglomerans (Beijerinck) Gavini et al. deficient in chitinolytic activity were unable to protect cotton (Gossypium barbardense L.) and expression of the chiA gene for endochitinase in Escherichia coli (Migula) Castellani & Chalmers allowed the transformed strain to inhibit R. solani on cotton seedlings. Similar techniques involving Tn5 insertion mutants and subsequent complementation demonstrated that biocontrol of Pythium ultimum in the rhizosphere of sugar beet by Stenotrophomonas maltophila (Hugh) Palleroni & Bradbury W81 was due to the production of extracellular protease (Dunne et al., 1997).
Induced resistance
Perhaps the greatest growth area in biocontrol in the last few years has been concerned with induced resistance defined as ‘the process of active resistance dependent on the host plant's physical or chemical barriers, activated by biotic or abiotic agents (inducing agents)’ (Kloepper et al., 1992). This has come about through the synergistic interaction of microbiologists, plant pathologists and plant scientists armed with an appropriate battery of molecular tools. The effect had previously often been overlooked through inadequate techniques or controls as well as the biocontrol agent exhibiting other modes of action at the same time. Most work has focused on the systemic resistance induced by non-pathogenic rhizosphere-colonizing Bacillus and Pseudomonas species in systems where the inducing bacteria and the challenging pathogen remained spatially separate for the duration of the experiment, and no direct interaction between the bacteria and pathogen was possible (Sticher et al., 1997; van Loon, 1997). Such split root or spatial root inoculation experiments were used to demonstrate the phenomenon in radish (Raphanus sativus L.) and Arabidopsis against Fusarium oxysporum (Leeman et al., 1996a; van Wees et al., 1997) and in cucumber (Cucumis sativus L.) against Pythium aphanidermatum (Edson) Fitzp. (Chen et al., 1998). Various combinations of timing and position have indicated that induced resistance also occurs in carnation (Dianthus caryophyllus L.) (van Peer et al., 1991), tobacco (Nicotiana tabacum L.) (Maurhaufer et al., 1994) and tomato (Lycopersicon esculentum Mill.) (Duijff et al., 1997). Bacteria differ in ability to induce resistance, with some being active on some plant species and not others; variation in inducibility also exists within plant species (van Loon, 1997). The full range of inducing moieties produced by bacteria is probably not yet known, but lipopolysaccharides (Leeman et al., 1995) and siderophores (Métraux et al., 1990; Leeman et al., 1996b) are clearly indicated.
The definition of induced resistance suggested by Kloepper et al. covered both biotic and abiotic inducers (Kloepper et al., 1992). Although the phenotypic effects of root inoculation with bacteria may be similar to treatment with abiotic agents or micro-organisms that cause localized damage, the biochemical and mechanistic changes appear to be subtly different. This has resulted in the term induced systemic resistance (ISR) for bacterially-induced resistance and systemic acquired resistance for the other forms (Pieterse et al., 1996). The major differences are that pathogenesis-related (PR) proteins such as chitinases, ß-1,3-glucanases, proteinase inhibitors and one or two other rarer types, are not universally associated with bacterially induced resistance (Hoffland et al., 1995) and salicylic acid (a known inducer of SAR) is not always involved in expression of ISR, but this is dependent on bacterial strain and host plant involved (Pieterse et al., 1996; de Meyer et al., 1999; Chen et al., 1999). Ethylene responsiveness may also be required at the site of inoculation of the inducing bacteria for ISR to occur (Knoester et al., 1999).
Changes that have been observed in plant roots exhibiting ISR include: (1) strengthening of epidermal and cortical cell walls and deposition of newly formed barriers beyond infection sites including callose, lignin and phenolics (Benhamou et al., 1996a, b, c, 2000; Duijff et al., 1997; Jetiyanon et al., 1997; M'Piga et al., 1997); (2) increased levels of enzymes such as chitinase, peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase (M'Piga et al., 1997; Chen et al., 2000); (3) enhanced phytoalexin production (van Peer et al., 1991; Ongena et al., 1999); (4) enhanced expression of stress-related genes (Timmusk and Wagner, 1999). However, not all of these biochemical changes are found in all bacterial–plant combinations (Steijl et al., 1999). Similarly, the ability of bacteria to colonize the internal tissue of the roots has been considered to be an important feature in many of the bacterial–root interactions involving ISR, but is not a constant feature of them all (Steijl et al., 1999).
Plant growth-promoting rhizobacteria (PGPR)
The concept of PGPR is now well established (Bashan, 1998; Shishido and Chanway, 1999) and so some consideration of the relationship of PGPRs to biocontrol is worthwhile. PGPR increase plant growth indirectly either by the suppression of well-known diseases caused by major pathogens or by reducing the deleterious effects of minor pathogens (micro-organisms which reduce plant growth but without obvious symptoms). Most of the bacteria discussed so far in this review fall into this category of PGPR. Alternatively, PGPR may increase plant growth in other ways, for example, by associative N2 fixation (Hong et al., 1991), solubilizing nutrients such as P (Whitelaw, 2000), promoting mycorrhizal function (Garbaye, 1994), regulating ethylene production in roots (Glick, 1995), releasing phytohormones (Arshad and Frankenberger, 1991; Beyeler et al., 1999), and decreasing heavy metal toxicity (Burd et al., 1998). It has been suggested that the two groups should be reclassified into biocontrol plant growth-promoting bacteria (biocontrol PGPB) and PGPB (Bashan and Holguin, 1998). To date this proposal does not seem to have been widely accepted, but it does highlight the need to consider the full ecological interactions taking place following application of bacteria to seeds and roots that lead to plant growth promotion. It is also important to remember that deleterious rhizobacteria that inhibit plant growth are also known (Nehl et al., 1996) which can influence such interactions.
Irrespective of mode of action, a key feature of all PGPR is that they all colonize roots to some extent. In some cases this may involve specific attachment through, for example, pili, as with the attachment of Pseudomonas fluorescens 2-79 to the surface of wheat roots (Vesper, 1987). However, such specific attachment does not seem to be an absolute requirement for colonization (de Weger et al., 1995). Colonization may involve simply root surface development but, endophytic colonization of the root is also known, and the degree of endophytic colonization depends on bacterial strain and plant type. Endophytic growth in roots has been recorded with the PGPR Bacillus polymyxa (Prazmowski) Macé Pw-ZR and Pseudomonas fluorescens Sm3-RN on spruce (Picea glaucaxP. engelmannii) (Shishido et al., 1999), with the biocontrol strains of Bacillus sp. L324-92R12 and P. fluorescens 2-79RN10 on wheat (Kim et al., 1997) and with several that induce resistance such as Bacillus pumilus Meyer & Gottheil SE34 and P. fluorescens 63-28 on pea (Pisum sativum L.) (Benhamou et al., 1996a, b; M'Piga et al., 1997), P. fluorescens CHA0 on tobacco (Troxler et al., 1997) and P. fluorescens WCS417r on tomato (Duijff et al., 1997). Large scale differences in spread within the plant may occur. Some, such as B. polymyxa Pw-2B, P. fluorescens SM3-RN, Bacillus sp. L324-92R12, and P. fluorescens 2-79RN10 spread from roots to aerial plant parts whereas others may not (Kim et al., 1997). Small scale differences are also known. For example, both B. pumilus SE34 and P. fluorescens 63-28 grow on the root surface and intercellarly in pea roots (Benhamou et al., 1996a, b; M'Piga et al., 1997) whereas surface growth, inter- and intra-cellular growth occurred with P. fluorescens WCS417r in tomato and P. fluorescens CHA0 in tobacco (Duijff et al., 1997; Troxler et al., 1997). These endophytic bacteria may be in a particularly advantageous ecological position in that they may be able to grow and compete on the root surface, but also may be capable of developing within the root, relatively protected from the competitive and high-stress environment of the soil. Indeed, many seeds, roots and tubers are normally colonized by endophytic bacteria (McInroy and Kloepper, 1995; Sturz et al., 1999). Any plant resistance encountered must be minimal, although, in many cases, sufficient to allow ISR to develop. The localized signalling between plant and bacteria within the root environment deserves further study. Certainly, use of mutants and promoter probe techniques are beginning to identify genes in bacteria that are important in colonization and these are often related to nutrient uptake (Bayliss et al., 1997; Roberts et al., 2000). Such nutrient uptake genes may also play a role in biocontrol by aiding the uptake and metabolism of nutrients that stimulate germination of pathogen propagules (Maloney et al., 1994).
The ability to colonize seeds is also an important feature for many bacterial biocontrol agents. Pseudomonas chlororaphis (Guignard & Sauvngeau) Bergey et al. MA342 is applied to cereal seeds to control many seed and soil-borne pathogens (Table 1
) and has been found to colonize specific areas of the seed coat (Tombolini et al., 1999). After inoculation, the bacteria were found under the seed glume (or husk), but after planting they were found to colonize the glume cells epiphytically. Bacterial aggregates were also found in the grooves formed by the base of the coleoptile and the scutellum, and near the embryo but never within it. In this case, the biocontrol bacteria co-located with the seed-borne pathogen Drechslera teres (Sacc.) Shoemaker providing biocontrol through the production of fungitoxic compounds. The spermosphere competence of this bacterium allowed biocontrol to take place. Microcolony or microaggregate production by bacteria has also been found on the grooves or cracks on the outer seed coat of sugar beet and cotton (Gossypium hirsutum L.) (Fukui et al., 1994; Hood et al., 1998) perhaps reflecting areas of increased nutrient availability or environmental protection.
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Fungal–protozoan interactions |
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TopAbstractIntroductionBacteria-bacterial pathogen...Bacteria-fungal pathogen...Fungal-protozoan interactionsFungal-bacterial pathogen...Fungal-fungal pathogen...Multiple microbial interactionsConclusions and future...References |
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The soil-borne protozoan Plasmodiophora brassicae Woronin is an ecologically obligate biotroph of brassicas causing clubroot disease which is characterized by proliferation of galls on infected roots. From a large-scale screening exercise, two isolates of the root-colonizing fungus Heteroconium chaetospira (Grove) Ellis were found to suppress clubroot on chinese cabbage (Brassica campestris L.) in non-sterile soil (Narisawa et al., 1998
). Hyphal growth occurred in the inner parts of the cortical tissues and into the root tips without causing any external symptoms on the plant and there was no sign of infection by P. brassicae. Further studies demonstrated that H. chaetospira infected epidermal cells from appressoria via infection pegs and, subsequently, intracellular hyphal growth occurred (Narisawa et al., 2000). However, the actual mechanism of the disease control observed in the field was unclear. Heteroconium chaetospira appears to form a mutualistic symbiosis with B. campestris in terms of disease control which is of interest as the Brassicacae family is largely non-mycorrhizal. In addition, H. chaetospira was found to colonize the roots of plants from eight families and may have a wide host range (Narisawa et al., 2000). Its ability to control diseases in these other plant families and the mechanisms involved deserves further study. |
Fungal–bacterial pathogen interactions |
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TopAbstractIntroductionBacteria-bacterial pathogen...Bacteria-fungal pathogen...Fungal-protozoan interactionsFungal-bacterial pathogen...Fungal-fungal pathogen...Multiple microbial interactionsConclusions and future...References |
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In the last few years there have been no clear examples of fungi used to control bacterial plant pathogens in the rhizosphere or spermosphere. The reasons for this are unclear but could perhaps indicate an area that deserves further research in the future.
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Fungal–fungal pathogen interactions |
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TopAbstractIntroductionBacteria-bacterial pathogen...Bacteria-fungal pathogen...Fungal-protozoan interactionsFungal-bacterial pathogen...Fungal-fungal pathogen...Multiple microbial interactionsConclusions and future...References |
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Interactions between biocontrol fungi and fungal plant pathogens continue to be the focus of a large number of researchers, on a par with work on bacterial–fungal plant pathogen interactions described earlier. However, there is an extra dimension in the quality of the interactions between fungi as biocontrol fungi have much greater potential than bacteria to grow and spread through soil and in the rhizosphere through possession of hyphal growth. Some recent examples of fungal–fungal interaction concerning biocontrol in the rhizosphere and spermosphere are given in Table 2
. There are a variety of fungal species and isolates that have been examined as biocontrol agents but Trichoderma species clearly dominate, perhaps reflecting their ease of growth and wide host range (Whipps and Lumsden, 2001). There has been an upsurge in interest in non-pathogenic Pythium species, particularly P. oligandrum Drechsler where additional modes of action have been determined recently, and a continued interest in well-established saprotrophic antagonists such as non-pathogenic Fusarium species, non-pathogenic binucleate Rhizoctonia isolates and Phialophora species, as well as mutualistic symbionts including mycorrhizal fungi such as Glomus intraradices Schenk & Smith. At least one novel biocontrol agent, Cladorrhinum foecundissimum Saccardo & Mardial, has been described. Numerous others are listed elsewhere (Whipps, 1997a). The most common pathogen targets are Pythium species, Fusarium species and Rhizoctonia solani reflecting their world-wide importance and perhaps their relative ease of control under protected cropping systems, although numerous other pathogens have been examined. Significantly, relatively few of the examples given in Table 2 involve studies in non-sterile soil or field conditions, with most carried out in soil-less conditions reflecting the need to keep the complexity of the system to a minimum in order to achieve reproducible control. Some specific examples of the modes of action found to occur in the rhizosphere and spermosphere during interactions between fungi and fungal plant pathogens are given below.
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Competition
There have been relatively few studies on competition for nutrients, space or infection sites between fungi in the rhizosphere and spermosphere recently. Competition for carbon, nitrogen and iron has been shown to be a mechanism associated with biocontrol or suppression of Fusarium wilt in several systems by non-pathogenic Fusarium and Trichoderma species (Mandeel and Baker, 1991
; Couteadier, 1992; Sivan and Chet, 1989) and competition for thiamine as a significant process in the control of Gaeumannomyces graminis var. tritici by a sterile red fungus in the rhizosphere of wheat (Shankar et al., 1994). Many studies have shown a relationship between increased colonization of the rhizosphere by a non-pathogen, associated subsequently, with disease suppression. This is well established for non-pathogenic strains of Fusarium oxysporum controlling pathogenic F. oxysporum on a variety of crop plants (Eparvier and Alabouvette, 1994; Postma and Rattink, 1991), hypovirulent or non-pathogenic binucleate strains of Rhizoctonia species to control pathogenic isolates of R. solani (Herr, 1995) and several fungi including Phialophora species, Gaeumannomyces graminis var. graminis and Idriella bolleyi (Sprague) von Arx as well as several non-sporulating fungi, to control G. graminis var. tritici (Deacon, 1974; Wong and Southwell, 1980; Kirk and Deacon, 1987; Shivanna et al., 1996). As just one example, I. bolleyi exploits the naturally senescing cortical cells of cereal roots during the early stages of the crop and outcompetes G. graminis var. tritici for infection sites and nutrients. Rapid production of spores, which are then carried down the root by water, continue the root colonization process and this is suggested to be a key feature in the establishment of the biocontrol agent on the root (Lascaris and Deacon, 1994; Allan et al., 1992; Douglas and Deacon, 1994).
Mycorrhizal fungi are also strong candidates for providing biocontrol through competition for space by virtue of their ecologically obligate association with roots. Ectomycorrhizal fungi because of their physical sheathing morphology may well occupy normal pathogen infection sites. Strangely, little work has been carried out to demonstrate this mechanism since it was first suggested (Marx, 1972) with most biocontrol interest focused on antibiotic production and induced resistance (Perrin, 1990; Duschesne, 1994). Similarly, arbuscular mycorrhizas also have potential to occupy space and infection sites on roots, but evidence suggests that biocontrol provided by arbuscular mycorrhizas relates more to induced resistance, improved plant growth and changes in root morphology rather than competition per se (Cordier et al., 1996; Norman et al., 1996; Mark and Cassells, 1996).
Antibiosis
Although production of antibiotics by fungi involved in biocontrol is a well-documented phenomenon (Howell, 1998; Sivasithamparam and Ghisalberti, 1998), there is little recent work clearly demonstrating production of antibiotics by fungi in the rhizosphere and spermosphere. Unlike the situation with biocontrol bacteria, there appear to be no detailed studies in biocontrol fungi of genes coding for antibiotic synthesis. Mutants with raised or decreased production of antibiotics are either natural spontaneous ones or generated by UV or chemical mutagenesis, with inherent problems of pleiotropic gene effects, rather than targeted gene disruption (Howell and Stipanovic, 1995; Graeme-Cook and Faull, 1991; Wilhite et al., 1994; Fravel and Roberts, 1991). Consequently, clear identification and understanding of the role of antibiotics in disease control lags far behind that in bacteria and needs to be addressed.
Antibiotic production by fungi exhibiting biocontrol activity has most commonly been reported for isolates of Trichoderma/Gliocladium (Howell, 1998) and Talaromyces flavus (Klöcker) Stolk & Samson (Kim et al., 1990; Fravel and Roberts, 1991) although in the last few years antibiotics have been at least partially characterized in Chaetomium globosum (Kunze) (Di Pietro et al., 1992). Minimedusa polyspora (J. W. Hotson) Weresub & Le Clair (Beale and Pitt, 1995) and Verticillium biguttatum Gams (Morris et al., 1995). Of particular interest are those studies where antibiotic production has a definite link to biocontrol. For example, Trichoderma (Gliocladium) virens (J. Miller, Giddens & Foster) von Arx comprises P and Q group strains, based on their antibiotic profiles (Howell, 1999). Strains of P group produce the antibiotic gliovirin which is active against Pythium ultimum but not against Rhizoctonia solani AG-4. Strains of the Q group produce the antibiotic gliotoxin which is very active against R. solani but less so against P. ultimum. In seedling bioassay tests, strains of the P group are more effective biocontrol agents of damping-off on cotton caused by Pythium, while those from the Q group are more effective as biocontrol agents of damping-off incited by R. solani (Howell, 1991; Howell et al., 1993). Thus there is strong circumstantial evidence for a role for antibiotics in biocontrol in this experimental system. This has been confirmed in a zinnia–Pythium system where T. virens G-20 incorporated into soil and potting mix resulted in disease suppression clearly associated with maximum accumulation of gliotoxin in the medium (Lumsden and Locke, 1989; Lumsden et al., 1992a, b). Gliotoxin minus mutants displayed only 54% of the Pythium disease suppressive activity in zinnia compared with the wild-type (Wilhite et al., 1994). Gliotoxin production by Trichoderma is also thought to be responsible for cytoplasmic leakage from R. solani observed directly on membranes in potting mix (Harris and Lumsden, 1997).
Production of hydrogen peroxide in the rhizosphere, catalysed by glucose oxidase from Talaromyces flavus is thought to be responsible for the biocontrol of Verticillium wilt caused by Verticillium dahliae Kleb. on eggplant (Solanum tuberosum L.) (Stosz et al., 1996). Purified glucose oxidase significantly reduced the growth rate of V. dahliae in the presence, but not the absence, of eggplant roots, suggesting that a supply of glucose from the roots was of major importance (Fravel and Roberts, 1991). Further, a single-spored variant, Tf-l-np, which produced 2% of the level of glucose oxidase activity of the wild-type did not control Verticillium wilt on eggplant in non-sterile field soil in a glasshouse experiment, whilst the wild-type provided significant control (Fravel and Roberts, 1991). Glucose oxidase also suppressed growth of V. dahliae in vitro and killed microsclerotia of V. dahliae in vitro and in soil.
Induced resistance
As with bacteria described earlier, the ability of fungi to induce resistance in plants and provide biocontrol has gradually been receiving more attention in the last few years. A considerable number of fungi previously described to provide biocontrol by mechanisms such as competition, antibiosis, mycoparasitism or direct growth promotion are now thought to provide control, at least in part, by this mechanism. These include saprotrophs such as non-pathogenic Fusarium isolates (Hervás et al., 1995; Larkin et al., 1996; Postma and Luttikholt, 1996; Fuchs et al., 1997, 1999; Duijff et al., 1998; Larkin and Fravel, 1999), Trichoderma species (Yedidia et al., 1999), Pythium oligandrum (Benhamou et al., 1997; Rey et al., 1998), non-pathogenic binucleate Rhizoctonia isolates (Poromarto et al., 1998; Xue et al., 1998; Jabaji-Hare et al., 1999), and Penicillium oxalicum Currie & Thom (de Cal et al., 1997) as well as mutualistic biotrophs such as mycorrhizal fungi (Volpin et al., 1995; Dugassa et al., 1996; Morandi, 1996; St Arnaud et al., 1997).
However, not all these studies used the strict criterion of spatial separation between application of the biocontrol fungus and the challenging pathogen to define induced resistance. Indeed, some simply measured changes in enzymes, PR-proteins or cell wall characteristics found to be induced in plants through SAR (described earlier) without involvement of a pathogen at all (Volpin et al., 1995; Morandi, 1996; Yedidia et al., 1999; Rey et al., 1998). Certainly with some mycorrhizal fungi it has been questioned whether the biochemical responses similar to induced resistance found following infection are of sufficient magnitude or quality, or too transient, to provide disease control (Dumas-Gaudot et al., 1996; Morandi, 1996; Mohr et al., 1998). Indeed, during some mycorrhizal syntheses there is little or no induced resistance response detected (Mohr et al., 1998). However, spatial or temporal separation experiments have indicated that increased levels of chitinases, ß-1,3 glucanases, ß-1,4 glucosidase, PR-1 protein, and peroxidase as well as cell wall appositions and phenolics may be associated with induced resistance due to fungi (Benhamou et al., 1997; Fuchs et al., 1997; Duijff et al., 1998; Xue et al., 1998; Jabaji-Hare et al., 1999). Nevertheless, more work is needed to identify the biochemical changes taking place in a larger number of fungal–plant combinations as not all these biochemical markers were found to be important in each system examined. Further, there appear to be differences in the quality of the induced resistance found between bacteria and on fungi on the same plant. For example, the suppression of Fusarium wilt (F. oxysporum f. sp. lycopersici) (Sacc.) Snyder & Hansen on tomato by Pseudomonas fluorescens WCS417r did not involve production of PR-1 protein and chitinases whereas that induced by F. oxysporum Fo47 did (Duijff et al., 1998). Again more work in this area is required to determine the extent of differences in induced resistance produced by bacteria and fungi.
The elicitors responsible for inducing resistance are not known in detail. Trichoderma species produce a 22 kDa xylanase that, when injected in plant tissues, will induce plant defence responses including K+, H+ and Ca2+ channelling, PR protein synthesis, ethylene biosynthesis, and glycosylation and fatty acylation of phytosterols (Bailey and Lumsden, 1998). However, whether such a system is active in roots exposed to Trichoderma is not known. Pectic oligogalacturonides released after hydrolysis by a non-pathogenic binucleate Rhizoctonia isolate may act as elicitors of defence responses in bean (Phaseolus vulgaris L.) (Jabaji-Hare et al., 1999).
Dose–response experiments involving non-pathogenic Fusarium species to control F. oxysporum on tomato have indicated that induced resistance is not an all or nothing response (Larkin and Fravel, 1999). By varying the level of inoculum of the inducing strain and the pathogenic isolate in soil, it was shown that some non-pathogenic isolates such as Fusarium CS-20 controlled Fusarium wilt effectively with antagonist levels of only 100 chlamydospores g-1 of soil (cgs) with pathogen densities of up to 105 cgs. In contrast, isolate Fo47 was effective only at antagonist densities of 104–105 cgs, regardless of pathogen density. Subsequent mathematical modelling provided evidence that CS-20 control was largely through induced resistance whereas Fo47 was active primarily through competition for nutrients (Larkin and Fravel, 1999). Similar dose–response effects were found with non-pathogenic isolate of F. oxysporum f. sp. ciceris (Padw.) Matuo & Sato and non-pathogenic isolates of F. oxysporum to control wilt of chickpea (Cicer arietinum L.) caused by pathogenic F. oxysporum f. sp. ciceris (Hervás et al., 1995). However, in addition, the plant genotype also seemed to influence the degree of resistance induced.
Mycoparasitism
There is a huge literature on the ability of fungi to parasitize spores, sclerotia, hyphae, and other fungal structures and many of these observations are linked with biocontrol (Jeffries and Young, 1994; van den Boogert and Deacon, 1994; Madsen and de Neergaard, 1999; Mischke, 1998; Al-Rawahi and Hancock, 1998; Davanlou et al., 1999). However, most of the microscopical observations concerning mycoparasitism have come from in vitro studies or sterile systems (Benhamou and Chet, 1996, 1997; Inbar et al., 1996; Cartwright et al., 1997; Benhamou et al., 1999; Davanlou et al., 1999) and examples clearly demonstrating mycoparasitism in the rhizosphere or spermosphere are rare (Lo et al., 1998). However, indirect population dynamic studies showed that mycelium of Rhizoctonia solani in the rhizosphere of potato was a prerequisite for development of the mycoparasite Verticillium biguttatum (van den Boogert and Velvis, 1992) and rhizosphere competence was strongly related to biocontrol in mycoparasite isolates of Trichoderma species (Sivan and Harman, 1991; Peterbauer et al., 1996; Thrane et al., 1997; Harman and Björkman, 1998).
The process involved in mycoparasitism may consist of sensing the host, followed by directed growth, contact, recognition, attachment, penetration, and exit. Although not all these features occur in every fungal–fungal interaction, the key factor is nutrient transfer from host to mycoparasite. Directed growth of hyphae of Trichoderma to hyphae of Rhizoctonia solani prior to penetration has often been observed (Chet et al., 1981) and the presence of host sclerotia have been shown to stimulate germination of conidia of Coniothyrium minitans Campbell (Whipps et al., 1991) and Sporidesmium sclerotivorum Uecker, Adams & Ayers (Mischke et al., 1995; Mischke and Adams, 1996). However, the factors involved in controlling directed growth in these systems have not been fully characterized. Similarly, the factors controlling recognition and binding between fungal host and parasite are not yet clear. This process may involve hydrophobic interactions or interactions between complementary molecules present on the surface of both the host and the mycoparasite such as between lectins and carbohydrates. With Trichoderma, there is good evidence of lectin production by both parasite and host Corticum (Sclerotium) rolfsii Curzi and involvement of lectins in the differentiation of mycoparasitism-related structures (Inbar and Chet, 1994; Neethling and Nevalainen, 1995). Recently, both hydrophobic characteristics and surface carbohydrate moieties have been investigated in the mycoparasite C. minitans as a prerequisite to examining the interaction with its host Sclerotinia sclerotiorum (Lib.) de Bary (Smith et al., 1998, 1999). Little is known of the signalling pathways following recognition of the host. However, preliminary evidence in Trichoderma harzianum indicates that the signal is transduced by heterotrimeric G proteins and mediated by cAMP (Omero et al., 1999).
As penetration or cell wall degradation are frequently observed during mycoparasitism, great emphasis has been placed on characterizing and cloning the extracellular enzymes such as ß-1,3 glucanases, chitinases, cellulases, and proteases produced by fungal biocontrol strains (Haran et al., 1996a; Peterbauer et al., 1996; Archambault et al., 1998; Deane et al., 1998; Vázquez-Garcidueñas et al., 1998). By manipulating their activity through construction of ‘overproducing’ mutants, enzyme-negative mutants or even transgenic plants expressing the enzyme, a role for their production in biocontrol has been implied. Several fungi have been examined in this way including Talaromyces flavus (Madi et al., 1997), but this type of work has essentially focused on Trichoderma species. For example, a series of transformants of T. longibrachiatum Rifai were constructed with extra copies of egl1 gene encoding the production of ß-1,4 endoglucanase (Migheli et al., 1998). When applied to cucumber seeds sown in Pythium ultimum-infested soil, the transformants with inducible or constitutive expression of egl1 were generally more suppressive than the wild type strain. In this case, it was suggested that P. ultimum was controlled through the action of ß-1,4 glucanase degrading the cellulose of the cell wall of the pathogen. Similarly, transformants of T. harzianum, overproducing proteinase encoded by prb1, provided up to a 5-fold increase in control of damping-off in cotton caused by Rhizoctonia solani (Flores et al., 1996). Interestingly, the best protection was provided by a strain which produced only an intermediate level of proteinase activity and it was suggested that very high levels of proteinase production might cause degradation of other enzymes which are important in the mycoparasitic process (Flores et al., 1996). In this regard, chitinases have received the greatest attention in mycoparasitism. Numerous studies have been made of ß-N-acetylhexosaminidase (EC 3.2.1.52) (which splits the chitin polymer into N-acetylglucosamine monomers in an exo-manner), endochitinase (EC 3.2.1.14) (which cleaves randomly at internal sites over the entire length of the chitin microfibril) and chitin 1,4-ß-chitobiosidase (exochitinases or chitobiosidases) (which releases diacetylchitobiose in a stepwise fashion such that no monosaccharides or oligosaccharides are formed) (Haran et al., 1996a; Schickler et al., 1998; Lorito, 1998). For example, transformants of T. harzianum CECT 2413 that over-expressed on 33 kDa endochitinase (chit33) were more effective in inhibiting the growth of Rhizoctonia solani in vitro compared with the wild type (Limón et al., 1999).
The combination of chitinases as well as other cell wall-degrading enzymes differ between species and strains (Lorito, 1998) and chitinases are differently expressed during mycoparasitism (Haran et al., 1996b; Mach et al., 1999; Zeilinger et al., 1999). For example, an N-acetylhexosaminidase (CHIT 102) was the first to be induced in T. harzianum T-Y, but as early as 12 h after contact with its host Sclerotium rolfsii, its activity diminished, while that of another N-acetylhexosaminidase increased (Haran et al. 1996b). In contrast, when Rhizoctonia solani was the host, CHIT 102 was stimulated along with three endochitinases within 12 h following contact but, as the interaction proc