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The Use of Beneficial Microbial Endophytes for Plant Biomass and Stress Tolerance Improvement.
Abstract and Figures
Endophytes are microorganisms that live within host plants for at least part of their life and do not cause apparent symptoms of diseases. In general, beneficial endophytes promote host plant growth, increase plant nutrient uptake, inhibit plant pathogen growth, reduce disease severity, and enhance tolerance to environmental stresses. As sustainable and renewable agricultural production (including current biofuel and bioenergy crops) increases in prominence, endophytic microorganisms will play important roles and offer environmentally-friendly methods to increase productivity while reducing chemical inputs. This review discusses various aspects of beneficial fungal and bacterial endophyte interactions with plants, including the physiological and molecular mechansims by which they benefit plant performance. We also discuss the potential for genetic modification of endophytes with useful genes, which could be used to impart additional traits following inoculation with the genetically engineered endophytes. Finally, we review US-issued patents over the past decade which relate to the use of fungal and bacterial endophytes for plant growth and stress tolerance improvement.
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... Endophytic bacteria were found in a variety of plant tissues of numerous plant species without causing disease symptoms, and in some cases exerting beneficial effects on their hosts (Lodewyckx et al. 2002). The endophytic bacteria can not only promote the growth of the host plant, but also help the host plant resist adversity (Mei and Flinn 2010;Cipollini et al. 2012). Thus, host plants have a better survival against biotic and abiotic challenges and competition via the action of beneficial endophytes. ...
... Thus, host plants have a better survival against biotic and abiotic challenges and competition via the action of beneficial endophytes. Notably, many studies have reported that endophytic bacteria can promote growth of host plants such as potato, tomato, wheat, rice, soybean and other plants (Sturz and Nowak 2000;Mei and Flinn 2010). Furthermore, endophytic bacterial compositions of leaves were dynamic, apart from bacterial competence to colonize plants as endophytes, the host plant and environmental factors can strongly influence the endophytic diversity of a particular plant (Pang et al. 2022). ...
Background and aims Endophytic bacteria of leaves play a crucial role in plant development, but how structure and co-occurrence networks within these communities respond to seed and soil microbial communities is unknown. Methods To address this, endophytic bacteria of pea (Pisum sativum L.) seedling leaves (SL) after growing 3 and flowering leaves (FL) after 6 weeks were studied using Illumina sequencing of 16S rRNA gene. Results Richness and diversity of endophytic bacteria of FL were similar to non-planted soil (NS) and were higher than SL and seeds (S), while those of SL endophytic bacteria were similar to S. PCoA analysis showed endophytic bacterial community was dynamic during the developmental stage. At the genus level, Bacteroides was enriched and dominant at SL and decreased at FL, whereas Pantoea, Helicobacter and Coriobacteriaceae_UCG_002 tended to be enriched and dominant at FL. Interestingly, the endophytic bacterial community at FL was similar to those at NS. Besides, S and SL networks both had higher modular structures than FL network. Among networks constructed by the combined dataset, the ecosystem of S – SL, S – FL and SL - FL had more microbial interaction than other ecosystems (SL – NS and FL - NS), while the SL – NS and FL - NS ecosystems showed the higher modularity indicating the more special and resistant to secondary extinction species existed. Conclusions In our present study, bacteria from seeds and soils play a critical role for endophytic bacterial community of pea leaves at its developmental stage.
... The response of endophytes and their mechanism of action relies on the coordination among the microbial community within the plant, a process known as "quorum sensing" [46]. Quorum sensing involves microorganisms interacting with plants based on signals and signal perception mechanisms adopted by both parties [47,48]. Microbes produce sensing signals that trigger intensive action. ...
Drought and salinity stresses significantly threaten global wheat productivity, limiting growth and reducing yields, thus endangering food security worldwide. These stresses disrupt physiological processes, impair photosynthesis, and hinder optimal growth and yield by diminishing water uptake, causing osmotic stress, ion toxicity, and oxidative stress. In response, various mitigation strategies have been explored, including breeding for stress-tolerant cultivars, improved irrigation techniques, and the application of exogenous osmoprotectants and soil amendments. Among these strategies, the emergence of rhizospheric and endophytic growth-promoting microorganisms has attracted significant attention. Therefore, a systematic review was undertaken to illustrate the role of endophytic bacteria in enhancing wheat tolerance to drought and salinity stresses. This review analyzes physiological mechanisms and research trends, identifies gaps, and discusses implications for sustainable agriculture. An analysis of the literature related to endophytic bacteria in wheat was conducted using databases of major publishers from 2004 to 2023. The review explores their mechanisms, such as phytohormone production and stress-responsive gene induction, emphasizing their contribution to plant growth and stress resilience. The current research trends indicate a growing interest in utilizing endophytic bacteria to mitigate these stresses in wheat cultivation, with studies focusing on understanding their physiological responses and interactions with wheat plants. Future research should concentrate on elucidating the role of endophytic bacteria in enhancing host plant tolerance to multiple stressors, as well as aspects like endophytic mechanism of action , endophytic lifestyle, and transmission pathways. Overall, endophytic bacteria offer promising avenues for sustainable agricultural practices, aiding in crop resilience and food security amid environmental challenges.
... To achieve sustainable agriculture, it is necessary to increase plant productivity and develop environmentally friendly biofertilizers to prevent plants from abiotic and biotic stresses (Noble and Ruaysoongnern 2010;Yang et al. 2009). In recent years, increasing attention has been paid to the mutual relationships between plants and mycorrhizal symbiosis related to stress tolerance and yield improvement (Mei and Flinn 2010). ...
Arbuscular mycorrhizal fungi (AMF) enhance plant tolerance to abiotic stresses like salinity and improve crop yield. However, their effects are variable, and the underlying cause of such variation remains largely unknown. This study aimed to assess how drought modified the effect of AMF on plant resistance to high calcium-saline stress. A pot experiment was performed to examine how AMF inoculation affects the growth, photosynthetic activity, nutrient uptake and carbon (C), nitrogen (N) and phosphorus (P) stoichiometric ratio (C:N:P) of maize under high calcium stress and contrasting water conditions. The results showed that high calcium stress significantly reduced mycorrhizal colonization, biomass accumulation, C assimilation rate and C:N stoichiometric ratio in plant tissues. Besides, the adverse effects of calcium stress on photosynthesis were exacerbated under drought. AMF inoculation profoundly alleviated such reductions under drought and saline stress. However, it barely affected maize performance when subjected to calcium stress under well-watered conditions. Moreover, watering changed AMF impact on nutrient allocation in plant tissues. Under well-watered conditions, AMF stimulated P accumulation in roots and plant growth, but did not induce leaf P accumulation proportional to C and N, resulting in increased leaf C:P and N:P ratios under high calcium stress. In contrast, AMF decreased N content and the N:P ratio in leaves under drought. Overall, AMF inoculation improved maize resistance to calcium-salt stress through enhanced photosynthesis and modulation of nutrient stoichiometry, particularly under water deficit conditions. These results highlighted the regulatory role of AMF in carbon assimilation and nutrient homeostasis under compound stresses, and provide significant guidance on the improvement of crop yield in saline and arid regions.
... Moreover, Fulchieri et al. (1993) reported that Gibs increase root hair density in root zones that are involved in water and nutrient uptake and enhance the growth of plants to tolerate pathogen attack. Shahzad et al. (2017) also confirmed that the Gibs-producing ability of B. amyloliquefaciens RWL-1 offers additional assistance to tomato plants, and the resulting improvement in tomato growth can induce resistance to F. oxysporum disease in tomato plants, suggesting interference with early infection processes that further resulted in limiting disease development (Mei and Flinn 2010). Therefore, it was assumed in this study that the B. amyloliquefaciens RaSh1 inoculation lessened the negative effects of A. alternata infection on pepper plants. ...
Plants encounter many biotic entities, such as fungi, bacteria, and nematodes, which induce biotic stress that disrupts normal metabolism and limits the growth and productivity of plants. Currently, the use of plant growth-promoting bacterial endophytes instead of synthetic fungicides is intriguingly eco-friendly. An in vitro and in vivo antagonistic approach using Bacillus amyloliquefaciens RaSh1 was used to mimic the pathogenic effect of Alternaria alternata. The results showed that B. amyloliquefaciens significantly inhibited pathogenic fungal growth in vitro. Further, Capsicum annuum L. (pepper plants) were grown and subjected to inoculation with B. amyloliquefaciens and infected with A. alternata, and then the growth attributes, photosynthetic pigments, physio-biochemical parameters, and the level of endogenous phytohormones were assessed. Under the pathogen attack, the main responses, such as plant length, total fresh and dry weights, total chlorophylls, and pigments, were reduced, accompanied by increases in H2O2. As well, infection of pepper with A. alternata caused downregulation in the plant hormonal system by significantly decreasing gibberellins, indole-3-acetic acid, abscisic acid, as well as cytokinin concentrations. Although, with B. amyloliquefaciens application, an enhancement in growth, photosynthetic pigments, proline, thiol content, phenylalanine ammonia-lyase, and peroxidase in pepper plant leaves appeared while the content of H2O2 decreased. Endogenous phytohormones were found to be upregulated in B. amyloliquefaciens-inoculated and diseased plants. The current study found that B. amyloliquefaciens RaSh1 rescued pepper plant growth by modulating antioxidant defense and regulating hormones, and could be used to control A. alternata in an environmentally friendly manner while maintaining sustainable agriculture and food security.
... In agriculture, these microorganisms influence host plants by promoting growth, increasing crop yield, nitrogen fixation, and phytohormone production. Moreover, they enhance plant tolerance to abiotic stresses like drought, salinity, and heavy metal toxicity (Rodriguez et al., 2009;Mei and Flinn, 2010). In forestry, endophytes facilitate faster tree growth, improved nutrient uptake, and resistance against pathogens. ...
Environmental changes pose significant challenges to sustainable agriculture, adversely affecting crop production and soil fertility. Factors such as drought, salinity, pathogens, and soil type exert their influence on the behavior of fodder crops by altering their morphological, biochemical, and molecular mechanisms, ultimately leading to reduced yields and productivity. Consequently, there is a pressing need to develop mitigation strategies aimed at enhancing the tolerance of forage crops to both biotic and abiotic stresses, addressing a critical challenge in sustaining their growth. In recent times, the use of biofertilizers has emerged as an environmentally friendly alternative to chemical fertilizers, holding promise for sustainable horticultural, agricultural, and forestry production systems. Notably, endophytic microorganisms play a pivotal role in promoting plant growth through direct or indirect mechanisms. Additionally, endophytic bacteria actively regulate gene expression responsible for the production of antioxidant enzymes, various phytohormones, siderophores, and ROS scavenging enzymes, all of which contribute to supporting the growth of host plants even in extreme environments. Consequently, there is a growing focus on understanding and validating the mechanisms through which beneficial plant endophytes interact to combat both biotic and abiotic stresses. This review emphasizes the potential of endophytes as biofertilizers, biocontrol agents, and contributors to the mitigation of abiotic and biotic stresses, all of which play crucial roles in maintaining the development of forage crops and soil fertility.
... In agriculture, these microorganisms influence host plants by promoting growth, increasing crop yield, nitrogen fixation, and phytohormone production. Moreover, they enhance plant tolerance to abiotic stresses like drought, salinity, and heavy metal toxicity (Rodriguez et al., 2009;Mei and Flinn, 2010). In forestry, endophytes facilitate faster tree growth, improved nutrient uptake, and resistance against pathogens. ...
... Numerous reports exist concerning the useful endophytic bacteria in the plant growth promotion like wheat, rice, canola, potato, tomato, and many more [12][13]. Most of these reports involve the possible growth promotion potential of endophytes isolated from the same plants. ...
... stina et al., 2013;Wang et al. 2014) or priming plant response to pathogens by induced systemic resistance (ISR) mechanism (Pieterse et al., 2014). Endophytes can be employed as bioinoculants in agriculture due to their ability to promote plant growth and fight disease, which will help the development of sustainable agricultural production methods (Mei and Flinn. 2010). ...
A class of endosymbiotic bacteria known as bacterial endophytes is
common among plants. A wide variety of bacterial taxa and host plants are
involved in the connection of endophytic bacteria with plants. Focusing on
the most recent findings acquired via metagenomic analysis, the present
study provides an overview of the taxonomic makeup of the bacterial
endophytes found in typical agricultural crops. The function and structure of
the soil and endophytic microbial populations are significantly influenced by
the endophytic microbiome, which is a component of the larger soil
microbial community and is susceptible to direct or indirect effects of
agricultural practices. In order to assure plant productivity and the quality of
agriculture products, it is crucial to utilize agricultural techniques that
preserve the natural variety of plant endophytic bacteria. On the other hand,
it has been demonstrated that the endophytic microbiome itself has several
impacts on the host plant, including the modification of pathways involved
in phytohormone signaling, metabolic activity and plant defense responses.
It has been shown that these effects could aid in the adaptation of plants to
biotic or abiotic stressors. Consequently, using endophytic bacteria to boost
disease resistance or crop performance under stress circumstances including
cold, drought, salt and heavy metal pollution offers a significant opportunity
for sustainable agricultural production.
... Henceforth, as a relatively simple and low cost alternative strategy, the use of plant growth promoting bacteria has been highlighted as a promising broad-spectrum means to improve plant growth (Yang et al., 2009). Endophytes are microorganisms which invade plant tissues and cause imperceptible and asymptomatic infections and improve plant growth by enhancing plant nutrient availability and imparting biotic and abiotic stress tolerance (Mei and Flinn, 2010). In recent years, plant microbiome, the entire collection of microorganisms associated with particular plant system has been studied and reported to benefit plant in a larger way. ...
Investigations were made to identify, screen and characterize rice apoplastic fluid endophytic bacterial strains for osmotic stress tolerance and plant growth promoting activity. These bacterial strains were identified phylogentically as Bacillus subtilis TSAC5, Bacillus subtilis TSAA2, Bacillus sp. R2AA10, Bacillus cereus R2AA7, Bacillus marisflavi TSAC7, Cupriavidus alkaliphilus TSAC1, Delftia sp. TSAC2, Janibacter melonis R2AA1, Microbacterium oleivorans R2AA6, Kocuria rosea R2AA5, Pseudomonas aeruginosa NAAI3 and Sphingobium yanoikuyae R2AI1. Of these 12 strains, eight were obtained from drought tolerant rice varieties such as Anna R4 and IR DRT 64 and four from normal rice variety CO51. These endophytes exhibited varied level of osmotic stress tolerance created using different concentrations of PEG 6000 (-1.5, -3.0, -4.0, -4.5, -5.0 and -5.5 MPa) under in vitro conditions in liquid and solid medium. Among the 12 strains, Cupriavidus alkaliphilus TSAC1, Delftia sp. TSAC2, Bacillus marisflavi TSAC7, Janibacter melonis R2AA1, Kocuria rosea R2AA5, Pseudomonas aeruginosa NAAI3 and Sphingobium yanoikuyae R2AI1 showed moisture stress tolerance up to -5.5 MPa. Further increase in moisture stress of -6.0 MPa inhibited growth of 12 tested strains. These strains also exhibited Indole acetic acid (IAA), gibberellic acid (GA3) and extracellular polysaccharide (EPS) production and also showed ACC deaminase (ACCD) activity. Maximum extracellular proline production was found in Bacillus marisflavi TSAC7 under both normal and water stressed conditions. Higher IAA productivity was registered in Janibacter melonis R2AA1 followed by Cupriavidus alkaliphilus TSAC1 and Sphingobium yanoikuyae R2AI1 in the absence of PEG 6000. In the presence of PEG 6000 Delftia sp. TSAC2 recorded maximum IAA productivity. Bacillus subtilis TSAA2 showed greater ACC deaminase activity. EPS production was in the order of Pseudomonas aeruginosa NAAI3 followed by Sphingobium yanoikuyae R2AI1. Sphingobium yanoikuyae R2AI1 produced maximum quantity of GA3 both under normal and water stressed condition. None of the tested strains indicated insoluble phosphate, silicate and zinc solubilisation.
The effects of arbuscular mycorrhizal (AM) fungi on plant development and root system morphology of different cultivars of Olea europaea were investigated. Rooted cuttings of olive cvs Frantoio, Moraiolo and Leccino at the beginning of the rooting phase, were inoculated with the AM endophyte Glomus mosseae. After six monthly growth increments, the percentage of root colonization and the root system morphology of the rooted cuttings were evaluated. AM inoculation enhanced growth of olive plants compared with the controls, inducing a different behaviour of the three olive cultivars colonized by G. mosseae. The fungal symbiont mainly increased the growth of the rooted cuttings of 'Moraiolo' and 'Frantoio', whereas 'Leccino' plants showed no significant differences from the controls. Staining of root samples of the cuttings indicated differences in the percentage of AM root colonization depending on cultivar. 'Moraiolo' root systems showed significantly less root length infected by Glomus mosseae compared with the other two cultivars. The morphometric analysis of olive roots demonstrated that colonization by AM fungi can significantly alter root system morphology. Colonization increased the intensity of branching on lateral orders in all cultivars and greatly increased the proportion of higher order laterals in the root system. The overall effect of mycorrhizas was, consequently, to increase lateral root frequency; giving rise to a more branched root system. Moreover the results show that such an increase in branching could be due in part to a better nutrition of mycorrhizal plants whereas mycorrhizal colonization and cultivar type greatly influenced the rooting pattern. These results show that mycorrhizal symbiosis can not only result in a significant increase in plant growth but can also alter olive root system morphology, positively affecting the establishment of plantlets.
Production of high quality, vigorous tissue culture-derived transplants requires efficient means to enhance their post-transplant ability for water management, photosynthesis and resistance to pathogens. Although there is increasing evidence that beneficial bacteria associated with plants are able to induce host resistance to abiotic and biotic stress, they have not been used in commercial micropropagation. We have documented that some of these beneficial bacteria can be co-cultured in vitro with plant explants and colonize the emerging plantlets. The bacterized plantlets appear more vigorous and withstand transplant stress better than non-inoculated controls. Their metabolism becomes pre-sensitised, or 'primed' to respond more quickly and to a higher degree to environmental challenges. A specific endophyte, Burkholderia phytofirmans strain PsJN, isolated from surface-sterilized onion roots, has conveyed improved growth and development to potato, tomato, sweet pepper, cucumber and grape plantlets. These primed plants have also exhibited improved performance under stress, including disease resistance to fungal pathogens. The phenotype of 'bacterized' plantlets in vitro is distinct (shorter, more branched and thicker roots, greater root and shoot biomass and more lignified and sturdier stems) and the clonal propagules or seedlings have improved survival and vigor after transplanting. However, the capacity for this beneficial plant-microbial interaction varies among cultivars and seems to be related to a population threshold of endophytic bacteria established in planta. To fully harness the potential benefits of bacterial endophytes for in vitro propagation we need to understand genetic determinants of the host-inoculant interaction under various environmental conditions. We have initiated such research using a unique population of sibling monoploid potatoes (derived by anther culture from a single heterozygous clone of adapted Solanum phureja and selected for high vigor) that differ for their ability to respond to PsJN. An ultimate goal of this research approach is the development of "value added" tissue culture propagules as a component of sustainable plant production system.
It has long been known that tissues of healthy plants can be colonized internally by microorganisms. The term "endophyte" is commonly used to describe such microorganisms. The best-characterized microbial endophytes are nonpathogenic fungi, for which much compelling evidence of plant/microbe mutualism has been provided. Some endophytic fungi are thought to produce compounds that render plant tissues less attractive to herbivores, while other strains may increase host plant drought resistance. In return, fungal endophytes are thought benefit from the comparatively nutrient rich, buffered environment inside plants. However, endophytic fungi comprise only part of the nonpathogenic microflora found naturally inside plant tissues. Bacterial populations exceeding 10(7) colony forming units (cfu) g(-1) plant matter have been reported within tissues of various plant species. Notwithstanding their discovery more than four decades ago, much less is known about bacterial endophytes compared to their fungal counterparts. Work with plant species of agricultural and horticultural importance indicates that some endophytic bacterial strains stimulate host plant growth by acting as biocontrol agents, either through direct antagonism of microbial pathogens or by inducing systemic resistance to disease-causing organisms. Other endophytic bacterial strains may protect crops from plant parasitic nematodes and insects. In Brazil, the nitrogen-fixing bacterial endophytes of sugarcane (Saccharum officinarun L.), Acetobacter diazotrophicus and Herbaspirillum spp., colonize internal root, stem and leaf tissues, and are thought to provide up to 80% of the host plant's nitrogen requirement. Other endophytic bacteria stimulate plant growth through mechanisms yet to be elucidated. In contrast to agricultural crop species, almost nothing is known about bacterial endophytes of trees. There have been occasional reports of endophytic bacteria in asymptomatic angiosperm and gymnosperm species, but little is known about their influence on plant growth. We have found that lodgepole pine (Pinus contorta var, latifolia Engelm.) and white x Engelmann hybrid spruce (Picea glauca x engelmannii) support bacterial endophyte populations naturally, and that such endophytes colonize internal root and stem tissues with up to 10(5) cfu g(-1) plant tissue. Furthermore, some of these strains have been found to promote gymnosperm seedling growth. While the precise mechanism by which these bacterial endophytes enhance tree seedling growth is not completely understood, initial results suggest that biocontrol of indigenous soil microorganisms that inhibit plant growth is at least partly involved. In addition, an endophytic Bacillus strain (Pw2), which was originally isolated from inside surface-sterilized pine root tissues, possesses nitrogenase activity and can colonize pine seedlings systemically after soil inoculation. These observations lead to the intriguing possibility that lodgepole pine harbors an endophytic nitrogen-fixing bacterial population similar to that of sugarcane, which would explain its ability to grow and even thrive, under nitrogen deficient conditions in the absence of significant rhizospheric nitrogen fixation. Bacterial endophytes may also be important in forest ecosystems by effectively increasing phenotypic plasticity of their long-lived tree hosts under variable or deleterious environmental conditions (e.g., during periods of drought, nutrient deprivation, or pathogen attack). Regardless of the mechanism(s) involved, bacterial endophytes appear to represent another type of mutualistic plant x microorganism symbiosis that warrants further study. In addition to the intriguing ecological questions regarding the diversity, evolution and effects on plant population biology of bacterial endophytes, it may be fruitful to investigate their possible practical applications in agriculture and forestry.
Screening of endophytic fungi which antagonize Colletotrichum musae, the cause of anthracnose disease, was carried out. The in vitro screening was studied by dual culture method. The inhibition due to fast competitive growth of Cordana sp. (KPP-3) and the antibiotic producing endophyte; Nodulisporium sp., reached a high percentage (90% C. musae inhibition). Spore germination assay showed 91% C. musae germination, while only 0.63 and 1.88% germinated in the conidial suspension of Cordana sp. and Nodulisporium sp., respectively. There was a significant difference between the Disease Severity Index (DSI) of banana fruits treated with Cordana sp. and those treated with Nodulisporium sp. The results presented in this research highlight the possibility of using endophytic fungi as biological control agents for anthracnose disease of banana.
Soil salinity is widely reported to be a major agricultural problem, particularly in irrigated agriculture, and research on salinity in plants has produced a vast literature. However, there are only a handful of instances where cultivars have been developed which are resistant to saline soils. Reasons for the lack of success in developing salt-resistant genotypes, and for the low impact that plant physiological research has made, are explored. We conclude that soil salinity has not yet become a sufficient agricultural problem, other than on a local scale, to make salt resistance a high priority objective for plant breeders. The limited success of simple selection, where this has been practised in breeding programs, can be accounted for by the fact that research has consistently shown salt resistance is a complex character controlled by a number of genes or groups of genes and involves a number of component traits which are likely to be quantitative in nature. We also conclude that the results of physiological research have been poorly marketed by physiologists and, understandably, have failed to impress plant breeders. We anticipate that the importance of salinity as a breeding objective will increase in the future. Our assessment of reports of the degradation of irrigation systems, together with projections of the future demands of irrigated agriculture, is that enhancing the salt resistance of at least some crops will be necessary. Salinity resistance will both help provide stability of yield in subsistence agriculture and, through moderating inputs, help limit salinisation in irrigation systems with inadequate drainage. It is emphasised that plant improvement and drainage engineering should be seen as partners and not alternatives. We conclude with a personal view of one way forward for developing salt-resistant genotypes, through the pyramiding of physiological characters.
Three methods of sampling xylem sap of maize roots were compared: sap bleeding from the stem cut just above the ground; sap bleeding from the cut tops of roots still undisturbed in the ground; and sap aspirated from excavated roots under reduced pressure. The bleeding saps were often unobtainable. When their composition was measured with time from cutting, the concentrations of the major solutes approximately doubled in 2 h. Aspirated sap was chosen as the most reliable sample of root xylem contents. Solute concentrations of the saps showed great variability between individual roots for all solutes, but on average the concentrations found (in µmol g-1 sap) were: total amino acids, 1.8; nitrate, 1.8; sugars (mainly sucrose), 5.4; total organic acids, 18.3. Individual amino acids also varied greatly between roots. Glutamine, aspartic acid and serine were generally most abundant. The principal organic acid found was malic, approximately 8 µmol g-1. From these analyses the ratios of carbon in the fractions (sugars : amino acids : organic acids) = (44 : 6 : 50). 14Carbon pulse fed to a leaf appeared in the root sap within 30 min, rose to a peak at 4-6 h, and declined slowly over a week. During all this time the neutral, cation and anion fractions were sensibly constant in the proportions 86 : 10 : 4. The 14C therefore did not move towards the equilibrium of 12C-compounds in the sap. It is argued that the results do not support a hypothesis of formation of amino carbon from recent assimilate and reduced nitrate in the roots and an export of this to the shoot in the transpiration stream.