Felix D. Dakora

Root exudates as mediators of mineral acquisition in low-nutrient environments

Plant developmental processes are controlled by internal signals that depend on the adequate supply of mineral nutrients by soil to roots. Thus, the availability of nutrient elements can be a major constraint to plant growth in many environments of the world, especially the tropics where soils are extremely low in nutrients. Plants take up most mineral nutrients through the rhizosphere where micro-organisms interact with plant products in root exudates. Plant root exudates consist of a complex mixture of organic acid anions, phytosiderophores, sugars, vitamins, amino acids, purines, nucleosides, inorganic ions (e.g. HCO3−, OH−, H+), gaseous molecules (CO2, H2), enzymes and root border cells which have major direct or indirect effects on the acquisition of mineral nutrients required for plant growth. Phenolics and aldonic acids exuded directly by roots of N2-fixing legumes serve as major signals to Rhizobiaceae bacteria which form root nodules where N2 is reduced to ammonia. Some of the same compounds affect development of mycorrhizal fungi that are crucial for phosphate uptake. Plants growing in low-nutrient environments also employ root exudates in ways other than as symbiotic signals to soil microbes involved in nutrient procurement. Extracellular enzymes release P from organic compounds, and several types of molecules increase iron availability through chelation. Organic acids from root exudates can solubilize unavailable soil Ca, Fe and Al phosphates. Plants growing on nitrate generally maintain electronic neutrality by releasing an excess of anions, including hydroxyl ions. Legumes, which can grow well without nitrate through the benefits of N2 reduction in the root nodules, must release a net excess of protons. These protons can markedly lower rhizosphere pH and decrease the availability of some mineral nutrients as well as the effective functioning of some soil bacteria, such as the rhizobial bacteria themselves. Thus, environments which are naturally very acidic can pose a challenge to nutrient acquisition by plant roots, and threaten the survival of many beneficial microbes including the roots themselves. A few plants such as Rooibos tea (Aspalathus linearis L.) actively modify their rhizosphere pH by extruding OH− and HCO3− to facilitate growth in low pH soils (pH 3 – 5). Our current understanding of how plants use root exudates to modify rhizosphere pH and the potential benefits associated with such processes are assessed in this review.

Contribution of legume nitrogen fixation to sustainable agriculture in Sub-Saharan Africa

Abstract Grain legumes fix about 15–210 kg N ha−1 seasonally in Africa, and therefore feature prominently in the cropping systems of traditional farmers. However, increased exploitation of this biological N is constrained by various environmental and nutritional factors, including the cropping patterns used. An evaluation of traditional cropping systems in Africa shows that crop rotation involving legume and cereal monocultures is by far more sustainable than intercropping, the most dominant cultural practice in the continent. Tree legumes also fix about 43–581 kg N ha−1 y−1, making their leaf prunings an important component of sustainability in agroforestry and alley cropping systems. In a single year, the prunings of Sesbania sesban can provide up to a hectare of cereal crop, up to 448 kg N, 31.4 kg P, 125 kg K, 114 kg Ca and 27.3 kg Mg, thus making the foliage of this legume the “ideal” fertilizer. Clearly, achieving sustainable yields in Sub-Saharan Africa would require a deeper understanding of how fixed N in legume residues is managed in the soil environment, in addition to expanding the use of neglected African food legumes, which exhibit considerable drought resistance and nitrate tolerance. In Africa, where soil moisture often limits yields, research on neglected, symbiotic native legumes with NO3− and drought-tolerant traits would constitute a sound basis for increased sustainable production.

Potential use of rhizobial bacteria as promoters of plant growth for increased yield in landraces of African cereal crops

Rhizobia form root nodules that fix nitrogen (N 2 ) in symbiotic legumes. Extending the ability of these bacteria to fix N 2 in non-legumes such as cereals would be a useful technology for increased crop yields among resource-poor farmers. Although some inoculation attempts have resulted in nodule formation in cereal plants, there was no evidence of N 2 fixation. However, because rhizobia naturally produce molecules (auxins, cytokinins, abscicic acids, lumichrome, rhiboflavin, lipo-chito-oligosaccharides and vitamins) that promote plant growth, their colonization and infection of cereal roots would be expected to increase plant development, and grain yield. We have used light, scanning, and transmission electron microscopy to show that roots of sorghum and millet landraces from Africa were easily infected by rhizobial isolates from five unrelated legume genera. With sorghum, in particular, plant growth and phosphorus (P) uptake were significantly increased by rhizobial inoculation, suggesting that field selection of suitable rhizobia/cereal combinations could increase yields and produce fodder for livestock production. Key Words: Rhizobia, N 2 fixation, plant growth, sorghum, millet African Journal of Biotechnology Vol.3(1) 2004: 1-7

Alfalfa (Medicago sativa L.) Root Exudates Contain Isoflavonoids in the Presence of Rhizobium meliloti

Root exudates of alfalfa (Medicago sativa L.) inoculated with symbiotic Rhizobium meliloti bacteria contained three isoflavonoids that were not found in exudates of uninoculated plants. Data from proton nuclear magnetic resonance, mass spectrometry, and ultraviolet-visible absorbance analyses indicated that root exudates of inoculated plants contained aglycone and glycoside forms of the phytoalexin medicarpin and a formononetin-7-O-(6″-O-malonylglycoside), a conjugated form of the medicarpin precursor formononetin. The medicarpin molecules did not induce nod gene transcription in R. meliloti, but the formononetin-7-O-(6″-O-malonylglycoside) induced nod genes regulated by both NodD1 and NodD2 proteins in R. meliloti. Hydrolysis of either the malonyl or the glycosyl linkage from the formononetin conjugate eliminated nod gene-inducing activity. The nod gene-inducing activity of crude root exudates was increased 200 and 65% upon inoculation with R. meliloti or R. leguminosarum bv phaseoli, respectively. When root exudate from uninoculated alfalfa was incubated with R. meliloti, high performance liquid chromatography analyses showed no evidence that bacterial metabolism produced medicarpin. These results indicate that alfalfa responds to symbiotic R. meliloti by exuding a phytoalexin normally elicited by pathogens and that the microsymbiont can use a precursor of the phytoalexin as a signal for inducing symbiotic nod genes.

Legume seed flavonoids and nitrogenous metabolites as signals and protectants in early seedling development

Flavonoids and nitrogenous metabolites such as alkaloids, terpenoids, peptides and amino acids are major components of plant seeds. Conjugated forms of these compounds are soluble in water, and therefore, are easily released as chemical signals following imbibition. Once in the soil, these metabolites are first in line to serve as eco-sensing signals for suitable rhizobia and arbuscular mycorrhizal (AM) fungal partners required for the establishment of symbiotic mutualisms. They may also serve as defence molecules against pathogens and insect pests, as well as playing a role in the control of parasitic members of the family Scrophulariaceae, especially Striga, a major plant pest of cereal crops in Africa. Seed metabolites such as flavonoids, alkaloids, terpenoids, peptides and amino acids define seedling growth and, ultimately, crop yields. Thus, an improvement in our understanding of seed chemistry would permit manipulation of these molecules for effective control of pathogens, insect pests, Striga and destructive weeds, as well as for enhanced acquisition of N and P via symbioses with soil rhizobia and AM fungi.

African legumes: a vital but under-utilized resource

Although nodulated legumes have been used by indigenous peoples in Africa for centuries, their full potential has never been realized. With modern technology there is scope for rapid improvement of both plant and microbial germplasm. This review gives examples of some recent developments in the form of case studies; these range from multipurpose human food crops, such as cowpea (Vigna unguiculata (L.) Walp.), through to beverages (teas) that are also income-generating such as rooibos (Aspalathus linearis (Burm. f.) R. Dahlgren, honeybush (Cyclopia Vent. spp.), and the widely used food additive gum arabic (Acacia senegal (L.) Willd.). These and other potential crops are well-adapted to the many different soil and climatic conditions of Africa, in particular, drought and low nutrients. All can nodulate and fix nitrogen, with varying degrees of effectiveness and using a range of bacterial symbionts. The further development of these and other species is essential, not only for African use, but also to retain the agricultural diversity that is essential for a changing world that is being increasingly dominated by a few crops such as soybean.

Influence of mycorrhizal associations on foliar δ15N values of legume and non-legume shrubs and trees in the fynbos of South Africa: Implications for estimating N2 fixation using the 15N natural abundance method

In this study, we examined the use of the 15N natural abundance method to quantify the percentage N derived from fixation of atmospheric N2 in honeybush (Cyclopia spp.) shrubs and trees in the fynbos, South Africa. Non-fixing shrubs and trees of similar phenology to the Cyclopia species were chosen as reference plants. These reference plants were selected to cover a range of mycorrhizal associations (ericoid mycorrhizal, arbuscular mycorrhizal and non-mycorrhizal). Isotopic analysis revealed a wide range of foliar δ15N values for the reference plants, including many very negative values. The marked differences in δ15N values were defined by the mycorrhizal status of the reference plant species, with the ericoid and arbuscular mycorrhizal plants showing lower foliar δ15N values relative to their non-mycorrhizal counterparts. In contrast, the δ15N values of the N2-fixing Cyclopia species were uniformly clustered around zero, from −0.11‰ to −1.43‰. These findings are consistent with the observation that mycorrhizal fungi discriminate against the heavier 15N isotope during transfer of N from the fungus to the host plant, leaving the latter depleted in 15N (i.e. with a more negative δ15N value). However, a major assumption of the 15N natural abundance method for estimating N2 fixation is that both legume and reference plant should have the same level of fractionation associated with N uptake. But, because mycorrhizal associations may strongly affect the level of fractionation during N uptake and transfer, the test legume should belong to the same mycorrhizal group as the chosen reference plant species. As shown in this study, if the mycorrhizal status of the legume and the reference plant differs, or cannot be assessed, then the 15N natural abundance technique cannot be used to quantitatively estimate N2 fixation.

Synthesis, release, and transmission of alfalfa signals to rhizobial symbionts

In addition to the flavonoids exuded by many legumes as signals to their rhizobial symbionts, alfalfa (Medicago sativa L.) releases two betaines, trigonelline and stachydrine, that induce nodulation (nod) genes inRhizobium meliloti. Experiments with14C-phenylalanine in the presence and absence of phenylalanine ammonia-lyase inhibitors show that exudation of flavonoidnod-gene inducers from alfalfa roots is linked closely to their concurrent synthesis. In contrast, flavonoid and betainenod-gene inducers are already present on mature seeds before they are released during germination. Alfalfa seeds and roots release structurally differentnod-gene-inducing signals in the absence of rhizobia. WhenR. meliloti is added to roots, medicarpin, a classical isoflavonoid phytoalexin normally elicited by pathogens, and anod-gene-inducing compound, formononetin-7-O-(6″-O-malonylglycoside), are exuded. Carbon flow through the phenylpropanoid pathway and into the flavonoid pathway via chalcone synthase is controlled by complexcis-acting sequences andtrans-acting factors which are not completely understood. Even less information is available on molecular regulation of the two other biosynthetic pathways that produce trigonelline and stachydrine. Presumably the three separate pathways for producingnod-gene inducers in some way protect the plant against fluctuations in the production or transmission of the two classes of signals. Factors influencing transmission of alfalfanod-gene inducers through soil are poorly defined, but solubility differences between hydrophobic flavonoids and hydrophilic betaines suggest that the diffusional traits of these molecules are not similar. Knowledge derived from studies of how legumes regulate rhizobial symbionts with natural plant products offers a basis for defining new fundamental concepts of rhizosphere ecology.

Nitrogen nutrition in nodulated field plants of the shrub tea legume Aspalathus linearis assessed using 15N natural abundance

Provision of N, P, and Ca to field plants of A. linearis markedly (P<0.05) increased growth and N nutrition in a very acidic nutrient-poor soil. Application of P and Ca promoted a significant increase in %N derived from fixation and amounts of N fixed compared to those receiving no nutrients. N2 fixation measured under field conditions ranged from 3.8 g N plant-1 in unfertilized control to 7.1 g N plant-1 in fertilized plants. Overall, about 85% increase in N2 fixation was observed with P supply. The high N2-fixing activity in P-treated plants was confirmed by their lower (more negative) ∂15N values. Age of plants also influenced growth and symbiotic activity as the ∂15N values, %N derived from fixation, and N fixed were lower in 1- and 2-year-old plants compared to 3-year-old. The contribution of symbiotic fixation in unfertilized A. linearis to the N economy of the ecosystem ranged from 105 kg N ha-1 in 1-year-old plants to 128 kg N ha-1 in 3-year-old plants, clearly indicating the remarkable adaptation of this symbiosis to the very nutrient-poor, low pH conditions of Cedarberg soils.

Effects of UV-B radiation on plant growth, symbiotic function and concentration of metabolites in three tropical grain legumes

Vigna unguiculata (L.) Walp. (cowpea), Glycine max (L.) Merr (soybean) and Phaseolus vulgaris (L.) (common bean) plants were exposed to UV-B radiation at above- and below-ambient levels, and their effects on growth, symbiotic performance and root concentration of metabolites were assessed. Moderately and highly elevated UV-B exposures averaging 32 and 62% above ambient had no effect on plant total dry matter, nodule number, nodule mass, nodule size, N fixed or root concentration of flavonoids, anthocyanins, soluble sugars and starch in the three species studied. However, N concentrations were markedly reduced in roots of G. max and P.vulgaris, and in leaves of P. vulgaris, which contrasted with the significant increase in stems and leaves of V.unguiculata. Below-ambient UV-B exposures averaging 22% of ambient also altered growth and metabolism of these legumes. Total plant dry matter, nodule number, nodule dry mass, N fixed and root starch concentrations in V.unguiculata decreased relative to both visible and UV-A radiation controls, whereas in G. max and P. vulgaris, these parameters were not altered. Root concentrations of flavonoids and anthocyanins in all species tested were also unchanged with below-ambient UV-B exposures. Taken together, growth and symbiotic function of these species remained unaltered with exposure to above-ambient UV-B, but differed in their response to below-ambient UV-B radiation.