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Research review Defining new roles for plant and rhizobial molecules in sole and mixed plant cultures involving symbiotic legumes

Author for correspondence: Felix D. Dakora Tel: +27 21 460 3878 Fax: +27 21 460 3692 Email: [email protected]

Felix D. Dakora Research Development, Cape Technikon Room 2.8 Administration Building, Keizersgracht PO Box 652, Cape Town 8000, South Africa

Received: 15 August 2002 Accepted: 19 December 2002 doi: 10.1046/j.1469-8137.2003.00725.x

Summary Key words: mixed plant cultures, rhizobia, legumes, Nod factors, nod gene inducers, lumichrome, riboflavin, pathogens.

The view that symbiotic legumes benefit companion and subsequent plant species in intercrop and rotation systems is well accepted. However, the major contributions made separately by legumes and their microsymbionts that do not relate to rootnodule N2 fixation have been largely ignored. Rhizobia (species of Rhizobium, Bradyrhizobium, Azorhizobium, Allorhizobium, Sinorhizobium and Mesorhizobium) produce chemical molecules that can influence plant development, including phytohormones, lipo-chito-oligosaccharide Nod factors, lumichrome, riboflavin and H2 evolved by nitrogenase. When present in soil, Nod factors can stimulate seed germination, promote plant growth and increase grain yields of legume and nonlegume crops, as well as stimulate increased photosynthetic rates following plant leaf spraying. Very low concentrations of lumichrome and H2 released by bacteroids also promote plant growth and increase biomass in a number of plant species grown under field and glasshouse conditions. Rhizobia are known to suppress the population of soil pathogens in agricultural and natural ecosystems and, in addition to forming nodule symbioses with rhizobia, the legume itself releases phenolics that can suppress pathogens and herbivores, solubilize nutrients, and promote growth of mutualistic microbes. Phytosiderophores and organic acid anions exuded by the host plant can further enhance mineral nutrition in the system. This review explores new insights into sole and mixed plant cultures with the aim of identifying novel roles for molecules of legume and microbial origin in natural and agricultural ecosystems. © New Phytologist (2003) 158: 39–49

Introduction Mixed cropping involving legumes, cereals, vegetables and tuber crops is a common practice in tropical Africa, Asia and South America. The management of both the crop

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component and the resident soil microbes such as root-nodule bacteria, arbuscular mycorrhizal (AM) fungi, and soil-borne pathogens is crucial for attaining higher yields in a sustainable manner. Effective N2 fixation and N contribution by root nodules is often viewed as the major role of symbiotic legumes

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Table 1 Molecules in legume root exudates that can affect symbiotic interactions in cropping systems Legume species Rhizobium symbiosis Alfalfa Cowpea

Common bean

Kersting’s bean

Soybean

Vetch

White clover

Bambara groundnut

Sesbania Lupin AM fungal symbiosis Alfalfa

White clover

Compound

Functional role

Reference

4,4′-Dihydroxy-2′-methoxychalcone 4′-7-Dihydroxyflavone liquiritigenin Daidzein Genistein Coumestrol Genistein-3-O-glucoside Eriodictyol Naringenin Daidzein Genistein Coumestrol Daidzein Genistein Coumestrol Isoliquiritigenin Genistein Genistein-7-O-glucoside Genistein-7-O-(6′′-O-malonylglucoside) Daidzein Daidzein-7-O-(6′′-O-malonylglucoside) Formononetin Biochanin A 3,5,7,3′-Tetrahydroxy-4′-methoxyflavanone 7,3′-Dihydroxy-4′-methoxyflavanone 2′,4′,4-Trihydroxychalcone 4′,4-Dihydroxy-2′-methoxychalcone Naringenin Liquiritigenin 7,4′-Dihydroxy-3′-methoxyflavanone 5,7,4′-Trihydroxy-3′-methoxyflavanone 5,7,3′-Trihydroxy-4′-methoxyflavanone 7,4′-Dihydroxyflavone Umbelliferone Formononetin Daidzein Genistein Coumestrol Liquiritigenin Erythronic acid Tetronic acid

nod gene inducer

Maxwell et al. (1989)

nod gene inducer

Dakora (2000)

nod gene inducer

Hungria et al. (1991)

4′,7-Dihydroxyflavone 4′,7-Dihydroxyflavanone Formononetin Formononetin Biochanin A

in cropping systems. As a result, successful nodulation of these leguminous species is regarded as the first step to achieving N benefit from these bacterial symbioses. The process of nodule formation in legumes involves the production by the plant of flavonoids, betaines and aldonic acids in its seed and root exudates (Table 1) as signals to the microbial symbiont (Phillips, 2000). In compatible associations, these compounds interact with NodD protein of the rhizobial cell and induce the expression of nodulation (nod )

Dakora et al. (1993b)

nod gene inducer

Dakora (2000)

nod gene inducer

Kape et al. (1992) Smit et al. (1992)

nod gene inhibitor

Djordjevic et al. (1987)

nod gene inducer

Recourt et al. (1991)

nod gene inducer nod gene inhibitor

Djordjevic et al. (1987)

nod gene inducer

Dakora & Muofhe (1996)

nod gene inducer nod gene inducer

Messens et al. (1991) Gagnon & Ibrahim (1998)

Hyphal growth promoter Spore germination inhibitor

Tsai & Phillips (1991) Tsai & Phillips (1991)

Hyphal growth promoter

Siqueira et al. (1991)

genes. The rhizobia, in turn, respond by releasing lipo-chitooligosaccharide Nod factors that cause morphological changes in legume root hairs, leading to infection thread formation, nodule development and symbiotic N2 fixation. The activity of rhizobial bacteroids inside the root nodules often results in large amounts of N2 fixed, ranging from 25 to 201 kg N ha−1 in tropical grain legumes (Dakora & Keya, 1997). This fixed N may benefit associated cereal crops (Eaglesham et al., 1981), and/or subsequent crops rotated

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with symbiotic legumes (Dakora et al., 1987). This explains why the inclusion of legumes in intercropping systems is largely seen in the context of fixed-N contribution to the system. Even with legume–cereal crop rotations, the overwhelming emphasis has been on symbiotic N benefit from the preceding legume crop, as well as on pest and pathogen control. Similarly, leguminous cover-crops, pasture legumes, and tree/shrub legumes, whether grown alone or in agroforestry systems, fix large amounts of N2 in tropical environments, and are also valued mainly for their contribution of N to nonleguminous crop species. It is clear from these considerations that our current understanding of sole and mixed cropping systems has been limited by earlier lack of interest in defining the various mechanisms that underlie microbial benefits to plants, and vice versa. So far, the beneficial effects of microorganisms on plants have generally been attributed either to suppression of pathogens or to plant hormones derived from bacteria. However, we now know that new active molecules released by legumes and other crop species can also impact positively on microbial growth and function, suggesting that these molecules are probably not unique to only legumes and their rhizobial partners. This review examines sole and mixed plant cultures involving symbiotic legumes with the aim of highlighting (1) recent discoveries of plant and bacterial molecules that are active under controlled conditions, and (2) the need to assess the extent of their function in natural and agricultural environments.

Rhizobial molecules as mediators of nutrient supply in plant cultures In addition to their involvement in the partnership that leads to symbiotic N2 fixation in nodules, rhizobia and legumes individually play other important roles in mixed cropping systems. These have remained largely unnoticed in the literature. Unlike rhizobial bacteroids, which reside inside root nodules and depend on the host plant for organic solutes and mineral nutrition, soil rhizobia have to obtain their nutrients directly from the environment. Consequently, the latter have evolved mechanisms that aid nutrient acquisition, especially in low-nutrient soils. Most rhizobial strains produce siderophores, indole acetic acid (IAA), and organic acids in culture media Antoun et al., 1998). As with plants growing in low nutrient environments (Dakora & Phillips, 2002), rhizobia use these exuded compounds to enhance mineral nutrition. Siderophores are used to mobilize iron (Fe) (Plessner et al., 1993), whereas organic acids solubilize phosphorus (P) and manganese (Mn). There is also evidence that plant roots in a mixed cropping system can benefit directly from this pool of bacterially solubilized nutrients (Loper & Buyer, 1991). Field studies (Howell, 1987) have shown that inoculating groundnut cv. Florunner with different rhizobial strains stimulated greater accumulation of calcium (Ca), P, magnesium (Mg), potassium (K), zinc (Zn) and other


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nutrient elements in seeds and nodules relative to that in uninoculated controls. Other bacteria such as the associative and endophytic microbes can also mobilize nutrients (e.g. P, Mg, Ca, Fe, etc.) that are not easily accessed at adequate rates by plants. A recent study by Riggs et al. (2001) showed significant yield increases in N-fertilized maize inoculated with the diazotrophic endophytes Klebsiella pneumoniae 342 and Herbaspirillum seropedicae Z152, possibly owing to enhanced mineral nutrition and promotion of plant growth. Although still hotly debated (McCully, 2001), various studies suggest that rhizobia and other microbes can enter roots of nonleguminous host plant species via cracks or at points of lateral root emergence (Spencer et al., 1994; de Bruijn et al., 1995; Reddy et al., 1997; Yanni et al., 2001) and establish themselves as endophytes in the xylem and intercellular spaces of these plants (Gough et al., 1997; O’Callaghan et al., 1997; Reddy et al., 1997). By contrast, McCully (2001) has argued strongly against the suitability of some of these niches for endophytic colonization. Whatever the case, rhizobia release growth-promoting molecules such as IAA, gibberellins and cytokinins (Phillips & Torrey, 1970, 1972; Dart, 1974; Lynch & Clark, 1984; Loper & Schroth, 1986; Law & Strijdom, 1989). Whether present in the rhizosphere as molecules from bacterial saprophytes or present in plant tissues as products released by endophytes, these compounds can massively proliferate root hair production (Yanni et al., 2001) and thus enhance the root’s absorptive capacity and nutrient uptake in both legume and nonlegume components of cropping systems. However, the effects of these phytohormones on root hair formation and nutrient uptake, remain to be tested experimentally under field conditions.

Rhizobial molecules as promoters of plant growth During nodule formation, rhizobia produce complex lipochito-oligosaccharide Nod factors as signals to the legume host. These, and other compounds, also serve as promoters of plant growth (Table 2). Recently, Zhang & Smith (2001) reported that imbibing plant seeds in solutions containing Nod factors stimulated germination and seedling development in eight angiosperm families. The application of Nod factors at 10−7 M or 10−9 M was found to increase soybean root mass by 7–16%, and root length by 34–44% (Smith et al., 2002). This stimulation in plant growth is consistent with the report that plant morphogenesis is regulated by lipo-chitooligosaccharide Nod factors (Spaink, 1996), compounds that have the ability to restore or resume cell division and embryogenesis in plant mutants and somatic embryo cultures (De Jong et al., 1993; Egertsdotter & von Arnold, 1998) in the absence of auxins and cytokinins (Daychok et al., 2000). In addition, spraying leaves of field plants with submicromolar concentrations (10−6, 10−8 or 10−10 M) of Nod factors caused a 10–20% increase in the photosynthetic rates of soybean, common bean, maize, rice, canola, apple and grape plants

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Table 2 Rhizobial molecules with potential for promoting plant growth in cropping systems Rhizobial compounds

Functional role

Reference

Nod factors

Stimulate seed germination Promote seedling development Increase foliar photosynthetic rates Induce expression of flavonoid genes Stimulate root colonization by arbuscular mycorrhizal fungi Cause cell division and embryogenesis Stimulates seedling growth Stimulates root CO2 production Serves as vitamin for plants and bacteria Promotes soil microbial populations Stimulates diversity of soil fauna Increases deposition of C in soil

Zhang & Smith (2001) Smith et al. (2002)

Lumichrome Riboflavin N2ase-linked H2

(Smith et al., 2001). This increase in photosynthate production led to an increase in grain yield of about 40% in field-grown soybean plants (Smith et al., 2001). An earlier study (Imsande, 1989) showed that inoculation of soybean at constant N supply to the plants with Bradyrhizobium japonicum increased photosynthesis, an effect that might be due to Nod factors released by the microsymbiont. However, more studies are needed to show that root application of Nod factors can promote photosynthesis in plant leaves. Although we still do not understand the mechanisms involved, the observations by Smith et al. (2001) imply that soil populations of rhizobia can release Nod factors that promote seed germination and seedling development in legumes while they simultaneously influence nodule formation. Nonlegume components of cropping systems such as maize and cotton can also benefit from stimulation of seed germination and early seedling development, especially at low temperatures. Phenolic compounds are known to also induce overexpression of Nod factors in rhizobia. In sole and mixed cropping systems, plant root exudation and decomposing residues can provide abundant phenolic nod-gene inducers (Table 1). High concentrations of these molecules in the soil can, in turn, cause an overproduction of bacterial Nod factors around roots, with the potential for stimulating plant growth even further, as observed by Smith et al. (2001). However, accumulation of Nod factors in the rhizosphere could also induce the expression of genes involved in flavonoid biosythesis such as those encoding phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase (Savoure et al., 1994; Spaink & Lugtenberg, 1994). This can, in turn, lead to increased phytoalexin accumulation and protection of plants against pathogens (Dakora & Phillips, 1996), or result in the release of greater concentrations of phenolic nod gene inducers by symbiotic legumes (Schmidt et al., 1994) with further promotion of rhizobial Nod factor production. Interestingly, rhizobial Nod factors also appear to play a major role in AM establishment since the application of low concentrations (10−9 M) of the bacterial metabolite was found to promote colonization of both nodulating and nonnodulating plant

Spaink & Lugtenberg (1994) Xie et al. (1995) Spaink (1996) Phillips et al. (1999) West & Wilson (1938) Dong & Layzell (2001)

roots by AM fungi (Xie et al., 1995). So, indirectly, rhizobial secretion of Nod factors could enhance AM fungal symbiosis with many potential benefits for the cropping system. Clearly, the role of rhizobia in cultural systems is far more complex than simply to participate in symbiotic N2 fixation in legumes. In addition to Nod factors, rhizobia produce riboflavin (Table 2; West & Wilson, 1938; Phillips et al., 1999), a vitamin that is also easily converted enzymatically or photochemically into lumichrome (Yagi, 1962; Yanagita & Foster, 1956). Recent evidence shows that lumichrome is actively produced by rhizobial cells in culture (Phillips et al., 1999), and stimulates CO2 production by alfalfa (lucerne) roots (Table 2), which, in turn, promotes rhizobial growth and accumulation of plant biomass (Phillips et al., 1999). Applying purified lumichrome to soybean, cowpea, sorghum and maize increased plant growth in the four species relative to the control (Dakora et al., 2002). However, the mechanism behind the promotion of growth by lumichrome is still unknown, although there is a proven requirement for exogenous CO2 for optimum growth of rhizobia (Lowe & Evans, 1962) in the same way that AM fungi require increased concentrations of CO2 for better growth (Becard & Piche, 1989; Becard et al., 1992). So, release of lumichrome-induced CO2 by the plant probably stimulates increased populations of rhizobia and mycorrhizal fungi in sole and mixed cropping systems. This would then have good prospects for improving symbiotic N and P nutrition of crop plants. Taken together, these findings suggest that field plants can potentially benefit from rhizobial exudation of lumichrome and riboflavin in both sole and mixed cropping systems. Rhizobial inoculation of plants has also been suggested to alleviate the effect of water stress in symbiotic legumes (Figueiredo et al., 1999). Although the mechanism underlying this observation is still unknown, it is possible that a rhizobial product such as abscisic acid (Phillips & Torrey, 1970, 1972) or lumichrome (Phillips et al., 1999) decreases leaf stomatal conductance and reduces water loss via transpiration in the leaves. Such a water-saving effect of bacterial molecules would be useful in sustaining N2 fixation in legumes during periods


Research review

of drought. However more experimentation is required to validate this hypothesis. Hydrogen gas (H2) is a major byproduct of nitrogenase activity during nodule function (see equation), and is thus produced by rhizobia. At peak growth of soybean, a hectare of N2-fixing plants can evolve about 5000 l of H2 per day from root nodules, accounting for about 5% of the crop’s daily net photosynthate (Dong & Layzell, 2001). N2ase N2 + 8e – + 8H+ + 16 ATP  → 2NH3 + 16 ADP + 16Pi + H2 In rhizobia with a hydrogenase uptake system (HUP+), H2 produced by nitrogenase is oxidized to feed electrons into an energy-generating electron transport chain. However, with HUP – strains, which commonly nodulate tropical grain legumes, H2 is evolved as gas into the soil environment. Although this was previously thought to be wasteful, recent studies (Dong & Layzell, 2001) show that pretreating field soil with H2 at concentrations similar to those around N2fixing root nodules can stimulate growth of soybean, barley, canola and spring wheat by 14, 18, 18 and 32%, respectively, relative to control plants. These growth responses were accompanied by marked increases in the number of plant tillers, number of heads and grain yield of field-grown barley and wheat. Growth promotion was associated with an increase in the population of soil bacteria (Table 2) and in their ability to oxidize nodule-evolved H2 (Dong & Layzell, 2002). The populations of springtails and insects that feed on bacteria also rose with the increase in microbial prey in H2treated soils. It is possible that some of these microbes were plant growth-promoting rhizobacteria, and that their increase in population would directly affect nutrient uptake and crop yields. The results of Dong & Layzell (2001, 2002) clearly indicate that H2 resulting from nitrogenase activity enhances soil biodiversity and can benefit both legume and nonlegume components in cropping systems. Their data also suggest that the improved growth of crop plants commonly observed in mixed cropping and/or crop rotations could stem partly from soil fertilization related to nitrogenase-linked H2 evolution. These findings show that rhizobia influence plant growth in more ways than simply through symbiotic N2 fixation.

Rhizobial control of plant pathogens Rhizobia are major biocontrol agents in natural and agricultural ecosystems. There is evidence that a strain of Bradyrhizobium japonicum can cause up to a 75% decrease in sporulation of Phytophthora megasperma, 65% in Pythium ultimum, 47% in Fusarium oxysporum and 35% in Ascochyta imperfecta (Tu, 1978, 1979). This suggests that a single strain of a root-nodule bacterium can have a suppressive effect on the soil population of a wide range of pathogens. Antoun et al. (1978) found 49 strains of Sinorhizobium meliloti that


Review

inhibited growth of F. oxysporum by up to 50%. Rhizobia isolated from root nodules of Acacia pulchella similarly decreased the survival of the zoospores of Phytophthora cinnamoni in vitro (Malajczuk et al., 1984), thus potentially providing bioprotection for the host plant. It is clear from these findings that rhizobia show great potential for use against plant diseases, and therefore deserve more attention in future studies of cropping systems. Field and glasshouse studies show that inoculating plants with rhizobia can be a cheap and effective method of controlling soil-borne pathogens in cropping systems. For example, inoculating soybean and common bean plants with their respective microsymbionts significantly decreased the severity of Phytophthora and Fusarium root rot in these species (Tu 1979; Buonassis et al., 1986). As with most parasitic interactions, the level of root rot decreased with increasing rhizobial numbers in soil (Tu 1979). Whether applied as seed dressing or soil drench, different rhizobial strains successfully protected field-grown soybean, mungbean, sunflower and okra plants from infection by the root-borne pathogens Macrophomina phaseolina, Rhizoctonia solani, and Fusarium species (Ehteshamul-Haque & Ghaffar, 1993). Taken together, these findings strongly indicate that, in nature, soil rhizobia act as biocontrol agents of plant diseases. Although Tu (1979) has suggested that rhizobia achieve this bioprotection by parasitizing the hyphal tips of the fungal pathogens and decreasing contact with the host plant cells, other mechanisms may exist. For example, the elicitation of isoflavonoid phytoalexins by rhizobial cells (Dakora et al., 1993a,b) or by their Nod factors (Savoure et al., 1994) can indirectly control pathogens in legumes. This effect of rhizobia may play a role in the control of viruses and bacterial pathogens, which, like fungi, also induce accumulation of isoflavonoid phytoalexins in host plant tissues (Dakora & Phillips, 1996), However, it is still unclear whether the same protection can be achieved in nonlegume hosts such as the cereals and vegetables in mixed cropping systems. There is some indication that, following infection, the AM fungus Glomus mosseae confers bioprotection against Phytophthora parasitica in roots of tomato plants (Cordier et al., 1998). This was shown by the presence of pathogenesis-related proteins in both mycorrhizal and nonmycorrhizal roots, suggesting that the tomato plant probably acquired both localized and systemic resistance against the pathogen. This observation parallels the finding that inoculating legumes with infective rhizobial cells (Dakora et al., 1993a,b), or with their Nod factors (Savoure et al., 1994), induces the synthesis and release of isoflavonoid phytoalexins that confer bioprotection to the plant. Interestingly, some rhizobia are themselves protected from the antimicrobial effects of induced phytoalexins by their nod-gene inducers (Parniske et al., 1991; Kape et al., 1992). Thus, pathogens are kept under control as rhizobia infect their host-plant roots. Although no specific studies have been done, it is possible that roothair infection by rhizobial

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bacteria also induces resistance against various pathogens of the host plant, as observed with G. mosseae (Cordier et al., 1998). Hopefully, future studies will provide direct evidence for this claim.

Role of natural endophytes in mixed plant cultures Several species of legumes, cereals, and tuber crops commonly found in the tropics, associate with N2-fixing diazotrophs that occur as natural endophytes of plants (Reynders & Vlassak, 1982; James et al., 1994; James, 2000; Dobbelaere et al., 2001). Interestingly, the benefits of these endophytes in mixed plant cultures are often ignored in functional analysis of the components, even though some of them make a significant contribution to the growth and N nutrition of cereals such as rice, maize, sugarcane, wheat, sorghum and barley (Dobbelaere et al., 2001; James, 2000). For example, NH4+releasing Azospirillum sp. selectively establish themselves intracellularly in paranodules (Christansen-Weniger & Vanderleyden, 1994) and contribute meaningfully to the N economy of the host plant. Inoculating cereal plants with these endophytes stimulates growth in maize and wheat, often leading to increased grain yields under field conditions (Fallik & Okon, 1996). Although the mechanism of stimulation remains unknown, Rodelas et al. (1993) have shown that microbial endophytes synthesize and release growth-promoting chemicals such as phytohormones (auxins, cytokinins and gibberellins), riboflavin and vitamins (thiamin, niacin and pantothenic acid), suggesting that these compounds may be the candidate molecules causing growth promotion in their host plants. This is in addition to the promotive effects of these endophytes on root hair proliferation with the associated potential for increased nutrient uptake by the host plant (Yanni et al., 2001). However, field experiments are needed to provide direct evidence for these roles of natural endophytes in cropping systems. Like cereals, legumes can also benefit from natural endophytes. Alfalfa root infection by the pathogenic endophyte Pseudomonas syringae pv. tabaci was found to double plant growth and enhance symbiotic N nutrition (Knight & Langston-Unkefer, 1988). Although the mechanism for growth promotion is still unknown, it is believed to stem from differential inhibition by bacterial toxins of glutamine synthetase (but not the nodule isoform) in the legume root. In addition, Burdman et al. (1997) observed an increase in nodulation and N2 fixation in common bean plants inoculated with Azospirillum brazilense alone, or co-inoculated with rhizobia. The increase in symbiotic performance of bean plants apparently resulted from an Azospirillum-induced increase in the release of nod gene inducers (Burdman et al., 1996) as well as increased production of lateral roots and root hairs (Burdman et al., 1998) that probably enhances rhizobial

infection and nodule formation. This might help explain the commonly observed increase in plant growth, seed yield and greater symbiotic benefits in several legume species inoculated with strains of A. brazilense (Burdman et al., 1998). Taken together, these findings imply that soil diazotrophs in cropping systems can enhance symbiotic function in legumes through stimulated release of nod gene inducers – aspects not often considered when assessing the role of biodiversity in mixed cropping systems. A number of studies (Al-Mallah et al., 1989, 1990; Spencer et al., 1994; Chabot et al., 1996; Noel et al., 1996; Reddy et al., 1997; Schloter et al., 1997; Yanni et al., 1997; Antoun et al., 1998) have explored the use of rhizobia as N2-fixing bacteria and as plant growth-promoting rhizobacteria for nonleguminous crop species such as wheat, maize, rice, potato, radish, oilseed rape and canola. It is now clear that rhizobia occur commonly as natural endophytes of many nonlegume plant species such as cotton and sweet corn (McInroy & Kloepper, 1995), Asian rice Oryza sativa (Yanni et al., 1997), African rice Oryza glaberrima, maize (Martinez-Romero et al., 2000), wheat (Biederbeck et al., 2000), and canola (Lupwayi et al., 2000). These discoveries were made either in field plants from mixed legume/cereal intercropping systems, or those involving long-term legume/cereal rotations. Yanni et al. (1997) have clearly established that these natural rhizobial endophytes promote plant growth and grain yields in various rice varieties in the Nile delta. However, such contributions by microbial endophytes are rarely mentioned in studies of sole or mixed plant cultures.

Legume molecules in mixed plant cultures In cropping systems, compounds in plant root and seed exudates perform various useful functions (Dakora, 1995) in addition to their role as communication molecules for nodule formation (Fig. 1; Table 2). Some phenolics from symbiotic legumes are known to promote growth of rhizobial bacteria in the rhizosphere (Fig. 1; Hartwig et al., 1991) and also to serve as chemoattractants that guide rhizobial cells to legume root hairs (Caetano-Anolles et al., 1988). In mixed cropping systems, and also in legume/cereal rotations, flavonoids secreted by root border cells (Hawes et al., 1998) and legume roots, or released from decomposing plant residues would, no doubt, enhance the soil pool of these compounds. This might result in promotion of growth of rhizobial cells and possibly increased supply of Nod factors for stimulation of plant growth. In addition, whether originating from soil or plants, these phenolic inducers can stimulate germination of fungal spores and growth of hyphae (Table 1; Tsai & Phillips, 1991; Xie et al., 1995), events important for the establishment of successful AM symbioses that may enhance N and P nutrition in legume and nonlegume crop plants. By contrast, exudation of isoflavones such as biochanin A and formononetin by legumes can inhibit germination of AM fungal spores and


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Fig. 1 Flavonoid molecules that promote germination of arbuscular mycorrhizal fungal spores/hyphal growth (A,C,D), induce nod genes in rhizobia (B,C,D) or stimulate growth of rhizobia (A).

hyphal development (Tsai & Phillips, 1991; Becard et al., 1992), and thus decrease the contribution of AM to the cropping system. High soil concentrations of certain isoflavones can also decrease seed germination and seedling development (Chang et al., 1969). Such compounds could either serve as suppressors of weeds, or as inhibitors of crop plant growth in cropping systems. Phenolics, phytosiderophores and carboxylic acids released as root exudates by legumes play a major role in the mineral nutrition of plants in low-nutrient environments (Dakora & Phillips, 2002). In addition to controlling soil pathogens, release of 2-(3′,5′-dihydroaxyphenyl)-5,6-dihydroxybenzofuran by alfalfa can enhance mobilization of P from Fe-bound P (Masaoka et al., 1993). In P-deficient soils, pigeonpea plants also use piscidic acid, and to a lesser extent, malonic acid and oxalic acid, to solubilize Fe-, Ca- and aluminum (Al)-bound P (Ae et al., 1990). Once mobilized, P and Fe then become available for uptake by the pigeonpea plant as well as by other associated plant species and microflora in the cropping system. In toxic Al soils, oxalic acid released by legume roots forms an Al–oxalate complex that renders the Al nontoxic to plants and mutualistic microbes in the cropping system (Ma et al., 1998). In that way, productivity of the cultural system is enhanced. Many plants have the ability to modify the pH of their rhizosphere (Hoffland et al., 1989, 1992; Raven et al., 1990; Degenhardt et al., 1998; Muofhe & Dakora, 2000) and enhance nutrient availability. This has the potential for promoting nutrient supply within crop rotations and to associated partners in mixed plant cultures. Preliminary data have shown that Cyclopia genistoides, a tea-producing legume indigenous to South Africa, can increase nutrient availability in its rhizosphere by up to 45–120% for P, 108–161% for K, 120–148% for Ca, 127–225% for Mg and by 117–250% for boron (B) compared with bulk nonrhizosphere soils (Dakora et al., 2000). Some species of lupins form cluster roots that release organic acid anions for solubilizing mineral


elements (Gilbert et al., 1999; Neumann et al., 1999) and create nutrient gradients between bulk, rhizosphere, and cluster root soils. The Rooibos tea (Aspalathus linearis) plant also forms cluster roots that exude organic acid anions. Thus, in mixed cultures, plants that lack this cluster root-forming trait can benefit directly from nutrients solubilized by cluster root exudates, just as they can similarly enhance their mineral nutrition in crop rotations with cluster root species. Furthermore, there are prospects that chemical molecules released by symbiotic legumes hold the key to the effective control of Striga, a parasitic weed that notoriously decreases grain yield of cereals and legumes in Africa. It appears that legumes first exude compounds that stimulate germination of Striga seeds, and then release a new set of molecules that kill the seedlings. This hypothesis is based on the observation that in legume/cereal rotations in Northern Ghana, the population of Striga hermonthica was decreased significantly from 17 plants m−2 in the cereal/cereal plot to 4, 5 and 6 plants m−2, respectively, with soybean, groundnut and cowpea as preceding crops (SARI, 1998), suggesting a molecular mechanism for the decrease in Striga density. The increase in grain yield of cereals after legumes ranged from 179% to 203%. These findings were confirmed by a recent study in northern Nigeria where the count of S. hermonthica seedlings was 2.22 per maize plant after sorghum as preceding crop, compared with 1.13 seedlings per maize plant after soybean, and 0.81 seedlings after P-fertilized soybean (Carsky et al., 2000). The observed increase in grain yield after soybean was attributed to symbiotic N supply and decreased parasitism by S. hermonthica. From these findings, symbiotic legumes seem to play a key role in the suppression of Striga and other parasitic weeds when rotated with cereals. However, the biological basis for this biocontrol of plant parasites by nodulated legumes, remains to be unraveled. It has been known for decades that plant defense against pathogens, nematodes, phytophagous insects and herbivores is based on the synthesis and accumulation of various

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Table 3 Isoflavonoid compounds from legume roots with potential for use as defense molecules against soilborne pests (insect larvae and pathogens) in cropping systems Legume species

Isoflavonoid

Defense role

Reference

Lotus pedunculatus Glycine max

Vestitol Glyceollin

Russell et al. (1978) Dakora & Phillips (1996)

Vigna unguiculata

Medicarpin

Phaseolus vulgaris

Phaseolin

Phaseolus lunatus Cajanus cajan

Coumestrol Cajanin

Lonchocarpus nicou

Rotenone

Derris malaccensis

Mundulea serica

Rotenone Deguelin Sumatrol Taxicarol Munduserone

Insect deterrent Insect deterrent Phytoalexin Insect deterrent Phytoalexin Insect deterrent Phytoalexin Nematicide Insect deterrent Phytoalexin Insecticide Phytoalexin Insecticide Phytoalexin

Pachyrrhizus erosus

Pachrrhizone

Neorautanenia pseudopachyrrhiza

Dolineone

Insecticide Phytoalexin Insecticide Phytoalexin Insecticide Phytoalexin

Dakora & Phillips (1996) Dakora & Phillips (1996) Rich et al. (1977) Dakora & Phillips (1996) Fukami & Nakajima (1971) Fukami & Nakajima (1971)

Fukami & Nakajima (1971) Fukami & Nakajima (1971) Fukami & Nakajima (1971)

phenolic compounds (Dakora, 1995). Simple and complex isoflavonoid molecules such as cajanin, glyceollin, rotenone, coumestrol, medicarpin, phaseolin, and phaseolidin are known to accumulate in tissues of tropical legumes, and act as phytoalexins, phytoanticipins, and nematicides against pests and herbivores (Table 3; Dakora & Phillips, 1996). When present in soil as a result of release from roots and/or from residue decomposition, these molecules can act against soilborne pathogens and root-feeding insects, as well as provide protection for crop plant species rotated with legumes. Although some flavonoid molecules are easily degraded by microbial activity in soil (Barz, 1970; Barz et al., 1970; Rao et al., 1991; Rao & Cooper, 1994), others persist long enough to induce transcription of nod genes in symbiotic rhizobia (Leon-Barrios et al., 1993). A number of isoflavones released by plants are known to decrease seed germination and reduce seedling development (Chang et al., 1969). Such compounds can usefully suppress weed species in cropping systems or, undesirably, inhibit crop plant growth in the field. Further research is needed on this aspect of isoflavonoid function in mixed plant cultures.

Conclusion The putative roles of known metabolites from plants and bacteria are summarized in Tables 1–3, and their potential effects on cropping systems are outlined in Figs 1 and 2. What is urgently needed now is field experimentation to provide a better understanding of the biological efficacy and

Fig. 2 A model of the interactive effects of plant and bacterial molecules in legume–cereal cropping systems. OM, organic matter; OAs, organic acid anions; AAs, amino acids; LCO, lipo-chito-oligosaccharide; AM, arbuscular mycorrhizas; BNF, biological N2 fixation.


Research review

functioning of these molecules in order for them to be properly managed for increased yields in cropping systems. However, to do so would require an interdisciplinary approach to studies of plant cultural systems. Future research on cereals and symbiotic legumes should therefore involve agronomists, biochemists, plant pathologists, plant physiologists, rhizobiologists, entomologists and soil scientists, as this stands a better chance of producing novel results that would transform current management of cropping systems for increased yields in the tropics.

Acknowledgements This study was funded with grants from the National Research Foundation, Pretoria, and the Cape Technikon.

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