Production of extracellular polysaccharides by a Rhizobium species from root nodules of Cajanus cajan

The ability of the Rhizobium sp., isolated from the root nodules of the leguminous pulse yielding shrub Cajanus cajan, to produce extracellular polysaccharides (EPS) was checked. A large amount of EPS (1, 128 μg/ml) was produced by the bacteria in yeast extract mannitol medium. Growth and EPS production started simultaneously, but the production reached its maximum level in the stationary phase of growth at 28 h. The EPS production by this Rhizobium sp. was much higher than by many other strains from nodules of Cajanus cajan which took a much longer time to reach maximum EPS production than this strain. The maximum EPS production (2,561 μg/ml) was obtained when the medium was supplemented with mannitol (1%), cetyl pyridinium chloride (2 μg/ml) and KNO₃ (0.2%), in which the production was increased by 276% compared to the control. The EPS production rose in the period up to 65 h with increased mannitol concentration. The EPS contained arabinose, xylose and rhamnose monomers. The possible role of rhizobial EPS production in root nodule symbiosis is discussed.     

Chemotaxis ofRhizobium sp. Towards root exudate ofCicer Arietinum L.

Summary Root exudate from seedlings ofCicer arietinum L. was collected in a chamber under aseptic conditions. The exudate was fractionated into anion, cation and neutral fractions. The anionic fraction was made up of galacturonic acid, gluconic acid, mannuronic acid and two unidentified compounds withR _f values 0.56 and 0.62. The cationic fraction contained alanine, arginine, aspartic acid, cystine, glycine, histidine, isoleucine, leucine, lysine and serine. The neutral fraction was made up of arabinose, galactose, glucose, ribose and xylose. The amino acids contributed to the bulk of the root exudate. The ratio of anionic, cationic and neutral fraction was 1∶7∶2. The crude root exudate was tested for its chemotactic ability using the capillary tube method. It was highly chemotactic for theRhizobium sp. The individual fractions and their various combinations were tested for chemotaxis. The chemotactic response of the Cicer strain of Rhizobium was least with anionic fraction most with cationic fraction and intermediate with neutral fraction. Maximum chemotactic response among the fractional combinations was obtained with all the three fractions and least with cationic plus neutral factions. Individual compounds constituting the various fractions were also tried for their ability to elicit chemotactic response. The organism exhibited maximum positive chemotactic response to histidine and negative response to alanine among the amino acids and to glucose and gluconic acid among the sugars and sugar acids.     

Chemotaxis in Rhizobium meliloti strain L5.30

We have found that Rhizobium meliloti strain L5.30 exhibits positive chemotaxis towards some amino acids, sugars, exudates and extracts from roots of legume plants. From the investigated compounds sugars were better chemo-attractants than amino acids, but legume root substances were the best ones. Positive chemotaxis towards legume root compounds was supported by clouds of R. meliloti cells observed at the surface of alfalfa roots. A large deletion, in the nodABC region of the symbiotic megaplasmid (Sym), did not eliminate rhizobial chemotaxis.     

Biochemical Changes in Root Exudate and Xylem Sap of Tomato Plants Infected with Meloidogyne incognita

Under two monoxenic culture techniques of growing plants (filter paper and silica sand cultures), sugar in root exudate from Meloidogyne incognita-infected tomato increased 133 to 836% over controls. In contrast, amino acids were moderately reduced 52 to 56%. Chromatographic analysis showed that galled root exudate contained three sugars, twelve amino acids, and three organic acids, whereas healthy root exudate contained four sugars, fifteen amino acids, and four organic acids. Polysaccharide was responsible for the large increase of sugars in galled root exudates. The concn and the absolute amount of total sugars in the infected plant xylem sap were greater than in healthy plant xylem sap up to 6 wk after inoculation, whereas amino acids were moderately lower than in controls throughout the test period. Chromatographic analysis showed that xylem sap from both healthy and infected plants at 4 wk after inoculation contained four sugars and five organic acids. We identified 18 and 17 amino acids in the healthy and infected plant xylem sap, respectively. The concn of sugar increased as the nematode inoculum increased at 2, 4 and 6 wk after inoculation. The amino acids in all samples from the infected plant moderately decreased with an increase of nematode inoculum. We suggest that changes in total sugars and amino acids, of infected plant xylem sap and root exudate are a probable mechanism by which tomato plants are predisposed to Fusarium wilt.     

Effect of phenolic compounds on symbiotic nitrogen fixation in pigeonpea(Cajanus cajan (L.) Millsp.)

The effect of different phenolic compounds:p-hydroxybenzoic acid, resorcinol and chlorogenic acid (mono-, di- and polyphenol) was studied on nodulation and related metabolic processes in pigeonpea (Cajanus cajan L. cv. Al-15). Nitrogenease activity, leghaemoglobin and ascorbic acid content of the nodules increased with the application of phenols. Phenols increase the contents of amino acids, proteins and total soluble carbohydrates in the nodules as reserve food materials.     

Salt tolerance of pigeon pea (Cajanus cajan (L.) Millsp.) at three growth stages

Salt tolerance of pigeon pea (Cajanus cajan (L.) Millsp.) was determined at three growth stages following observations by a number of workers that degree of salt tolerance of different crops varies with their ontogeny. The salt tolerance of three accessions, Local arhar, ICPL-151 and ICPL-850014 of pigeon pea was assessed at the germination, seedling and adult stages. There was no positive correlation between tolerance at the early growth stages and at the adult stage since no clear difference in salt tolerance of the three accessions was observed at the germination and the seedling stages, whereas accessions differed considerably at the adult stage. Although increasing salt concentrations adversely affected the growth of all three accessions, ICPL-151 was superior to the other two accessions in fresh and dry biomass, yield and yield components when tested at the adult stage. The tolerant accession, ICPL 151, accumulated significantly lower Na⁺and CI in shoots. By contrast it was higher in shoot and root K⁺, K/Na ratios, K vs Na selectivity, soluble sugars, free amino acids and proline compared with the other two accessions.     

Root exudates from lentil (Lens culinaris medic) seedlings in relation to wilt disease

Summary Root exudates from healthy and diseased lentil plants (Lens culinaris) have been investigated in relation to the wilt disease caused byFusarium oxysporum f.lentis. In all ten amino acids and five sugars have been detected. The spore germination of the pathogen in root exudates indicated that 21-days root exudate was inhibitory. Glycine and phenylalanine were detected in 21-days exudate and were found to have an inhibitory effect upon the germination of the spores of the pathogen which may partly explain the lesser disease incidence when plants of more than 3 weeks are inoculated with the pathogen.     

Symbiotic effectiveness of Hup+ Rhizobium,VAM fungi and phosphorus levels in relation to nitrogen fixation and plant growth ofCajanus cajan

Effect of hydrogen uptake positive (Hup⁺) strain ofRhizobium sp. (pigeon pea) and VAM fungus (Glomus fasciculatum) was studied on the symbiotic parameters of pigeon pea (Cajanus cajan) cv. AL-15 at various levels of phosphorus. The Hup⁺ Rhizobium strain showed more nodulation, plant biomass and plant nitrogen content than its Hup⁻ counterpart. VAM infection in pigeon pea roots helped in translocating phosphorus from the soil and improved nitrogen fixation. Similarly, addition of phosphorus was found to play a positive role in enhancing all these parameters. Dual inoculation of Hup⁺ Rhizobium strain and VAM significantly increased nodulation, nitrogenase activity, plant nitrogen and phosphorus content and plant biomass compared to single inoculation of either organism and dual inoculation with Hup⁻ and VAM fungus.     

Biochemical composition of stigmatic exudate of Eichhornia crassipes (Mart.) solms

The stigmatic exudate of Eichhornia crassipes (Mart.) Solms is moderately soluble in water, but is readily soluble in 80% ethanol. Soluble sugars, fructose, sucrose, total phenol, hydroxy phenol, free amino acids, soluble proteins and free fatty acids were found in the exudate. The exudate is devoid of organic acids and alkaloids, while proteins, soluble sugars, free fatty acids and free amino acids were the principal constituents. The stigma possesses detectable quantities of these compounds and shows a progressive increase in the amount of these substances up to the stage just prior to anthesis. The ultraviolet absorption profile of the stigmatic exudate in ethanol shows characteristic absorption peaks and bathochromatic shifts on addition of NaOH. The peaks probably correspond to those of simple phenolic compounds.     

Role of Amino Acids in Evolution of Ethylene and Methane, and Development of Microshoots in Cajanus Cajan

In pigeonpea (Cajanus cajan L.) shoot buds were induced when cotyledonary node explants were supplemented with benzylaminopurine (BAP; 2 mg dm^–3). When 0.1 mg dm^-3 BAP and 0.01 mg dm^-3 naphtahalene acetic acid were supplemented to the medium, the 34 – 35 % of induced buds developed to microshoots. By supplementing amino acids like proline, glutamine, asparagine and L-cysteine, shoot bud development to microshoots was enhanced at least by two fold. Amongst the amino acids proline gave maximum number of microshoots per explant. With increase in concentration of amino acids, fresh mass increased but microshoot number decreased. Also methane evolution was increased by addition of amino acids, and also in medium containing more of its nitrogen in the form of ammonia. Increased evolution of methane was accompanied by reduction in evolution of ethylene, and enhancement of efficiency of microshoot development.     

Physiology of behavioral mutants of Rhizobium meliloti: evidence for a dual chemotaxis pathway

Wild-type and nonchemotactic mutant strains of Rhizobium meliloti were tested for attraction to localized sites on alfalfa roots and for attraction to numerous small molecules, including sugars, amino acids, and two fractions derived from alfalfa root extracts. Four strains (carrying mutations che-6, che-11, che-12, and che-26) lost all responses and were classified as generally nonchemotactic mutants. One strain (carrying mutation che-7) lost responses to a group of structurally unrelated amino acids but retained all other responses and was classified as a putative sensory transducer mutant. Two strains (carrying mutations che-1 and che-3) lost responses to all the amino acids and sugars tested but retained normal responses to localized sites on roots and to the root fractions. These two mutant strains could not be classified according to the generally accepted model for a sensory pathway, derived from studies of enteric bacteria, and provided evidence for a dual chemotaxis pathway in R. meliloti.     

Spatial and temporal variations in phloem sap composition of plantation-grown Eucalyptus globulus

Spontaneous bleeding of sugar-rich sap from cambial-deep incisions in the bark of trunks was demonstrated for Eucalyptus globulus and other eucalypts across a range of localities and seasonal conditions in south-west Australia. High levels of sucrose and raffinose (up to 31% w/v total sugars) were present in the exudates, and upward and downward gradients in exudate sugar concentrations were recorded between samples obtained at different heights up trunks of E. globulus. The data indicated a phloem origin for the exudates, with source:sink pressure gradients driving translocation. Concentration ratios of sugars to amino acids were consistently lower in exudate from upper (distal) than basal regions of trunks, suggesting preferential partitioning of nitrogen upwards towards the trunk apex. A comparison of phloem and xylem sap composition from one plantation over a season showed nitrate in xylem but not phloem and substantial amounts of sodium, and high concentrations of chloride and sulphate relative to phosphate in xylem and phloem. Phloem sap sampled across a range of 29 contrasting plantations of E. globulus at peak stress (autumn) showed great inter-site variability in concentrations of amino acids, sulphur, sodium and certain trace elements and in C:N and Na:K ratios of sap. Carbon isotope ratios (δ¹³C) were strongly correlated with sugar concentrations of the sap samples from these and other plantations. Use of sap compositional attributes of phloem and δ¹³C values of translocated carbon is suggested for assessing the current nutritional condition and water status of E. globulus plantings. Received: 9 April 1998 / Accepted: 20 August 1998     

Studies on fast and slow growing Rhizobium spp. nodulating Cqjanus cajan and Cicer arietinum

Fast and slow growing Rhizobium spp. isolated from Cajanus cajan and Cicer arietinum were compared in terms of colony characteristics, utilisation of carbon sources, acid production, symbiotic effectiveness and nodulating competitiveness. Fast growing isolates from C. cajan and C. arietinum formed 3–6 mm diameter colonies on yeast-extract mannitol agar after 4 days and were unlike the slow growers which produced colonies of c. 1 mm diameter after 7–10 days at 28 °C. The fast growing Rhizobium spp. from C. cajan utilised a wider range of carbon sources than the slow growing isolates from this host. Fast and slow growing strains from C. arietinum were able to utilise most of the carbon sources tested suggesting that the slow growers possessed glycolytic pathways similar to those in other fast growing species of Rhizobium. In culture, slow growing isolates from C. cajan produced a near-neutral to alkaline reaction (pH 66·7-5) whereas the fast growers from this host and both fast and slow growing isolates from C. arietinum produced an acidic reaction (pH 4·4–5·6). These data are discussed in the context of Norris' (1965) evolutionary concept of the Leguminosae. Under glassshouse conditions, fast and slow growing strains isolated from C. cajan and C. arietinum were equally effective on their respective hosts. In competition with slow growing rhizobia, half of the fast growers formed more than 70% of the nodules on C. cajan grown in sand. In all but one instance similar results were obtained when plants were grown in soil. With C. arietinum grown in sand or soil, all fast growing isolates from this host formed more than 85% of the nodules in competition with slow growing strains.     

Effect of root exudates on the spore germiantion of rhizosphere and rhizoplane mycoflora of chilli (Capsicum annuum L.) cultivars

Summary From root exudates of three cultivars of chilli (Capsicum annuum L.) 12 amino acids and 7 sugars were detected. Methionine, d-1- phenylalanine, citrulline and d-xylose were detected only from the root exudates of resdistant cultivars. The root exudates of resistant variety inhibited spore germination of the pathogen (Fusarium oxysporum f. sp.capsici), but that of susceptible variety enhanced spore germiantion of the same. Spore germiantion of antagonistic fungi (Trichderma viride andAspergillus sydowi) was also influenced by the root exudates of resistant and susceptible varieties, but the influence was different.Spore germiantion of a number of rhizosphere fungi was studied and in general root exudate of susceptible cultivar enhanced spore germiantion of majority of fungi, but spore germination of antagonistic fungi against the pathogen was inhibited. However, root exudate of resistant cultivar stimulated spore germination of antagonistic fungi.     

Two rhizobacterial strains,individually and in interactions with Rhizobium sp., enhance fusarial wilt control,growth, and yield in pigeon pea

A Pseudomonas aeruginosa strain, RRLJ 04, and a Bacillus cereus strain, BS 03, were tested both individually and in combination with a Rhizobium strain, RH 2, for their ability to enhance plant growth and nodulation in pigeon pea (Cajanus cajan L.) under gnotobiotic, greenhouse and field conditions. Both of the rhizobacterial strains exhibited a positive effect on growth in terms of shoot height, root length, fresh and dry weight, nodulation and yield over the non-treated control. Co-inoculation of seeds with these strains and Rhizobium RH 2 also reduced the number of wilted plants, when grown in soil infested with Fusarium udum. Gnotobiotic studies confirmed that the suppression of wilt disease was due to the presence of the respective PGPR strains. Seed bacterization with drug-marked mutants of RRLJ 04 and BS 03 confirmed their ability to colonize and multiply along the roots. The results suggest that co-inoculation of these strains with Rhizobium strain RH 2 can be further exploited for enhanced growth, nodulation and yield in addition to control of fusarial wilt in pigeon pea.     

Evaluation of a novel tool for sampling root exudates from soil-grown plants compared to conventional techniques

The release of low molecular weight (LMW) organic compounds (e.g. organic acids, amino acids, sugars, etc.) by living plant roots significantly contributes to the development of chemical, physical as well as microbial rhizosphere gradients. Suitable and accurate sampling procedures are crucial for enhancing our understanding of the dynamics of related rhizosphere processes. Here we compare common sampling techniques with a novel tool for root exudate collection that allows non-destructive and repetitive sampling from soil-grown roots. Root exudates from Zea mays L. were collected using the following techniques: (i) hydroponic growth and sampling, (ii) soil growth and hydroponic sampling and (iii) rhizoboxes fitted with a novel in situ root exudate collecting tool. Furthermore, rhizosphere soil solution for the analysis of exudates and microbial metabolites was sampled using micro-suction cups (iv). The effect of different sampling solutions (deionised water and 0.5 mM CaCl₂) on organic acid and amino acid exudation patterns was also investigated. The novel exudate collecting tool was successfully tested for root exudate sampling. Results showed that particularly amino acid exudation rates were significantly affected by growth conditions and sampling procedures, while organic acid exudation patterns varied less across the different sampling setups. Despite qualitative and quantitative differences, exudation rates were in the same order of magnitude across the different sampling procedures. Soil solution concentrations obtained from micro-suction-cup sampling at defined distance to the root surface showed no distinct gradient, highlighting the importance of soil microorganisms in regulating the soil solution concentration of LMW C compounds either via microbial degradation or the release of microbial metabolites. The exudate collector offers new opportunities to assess root exudation rates and composition from soil-grown plants and thus enhances our knowledge of fundamental rhizosphere processes.     

Motility and chemotactic response of Pseudomonas fluorescens toward chemoattractants present in the exudate of Macrophomina phaseolina

Pseudomonas fluorescens strains (LAM1-hydrophilic) and (LAM2-hydrophobic) showed positive chemotaxis towards attractants (sugars, amino acids, polyols and organic acids) present in the exudate of Macrophomina phaseolina (a soilborne plant pathogenic fungus). The varied response of motility traits such as speed, rate of change in direction (RCDI) and net to gross displacement ratio (NGDR) was observed for different chemoattractants. Swimming speed of the strains was highest in 10-fold diluted exudate or 100–1000 μM strength of different attractants, but further dilutions significantly decreased the swimming speed (P = 0.05). Chemotactic response of P. fluorescens was positively correlated with swimming speed (P = 0.05; r = 0.76). Relative to control, the RCDI values decreased 1.5-fold in amino acids or sugars, and 1.2-fold in polyols or organic acids. With increase in swimming speed, the NGDR of both strains also increased, but the RCDI decreased. Both hydrophilic and hydrophobic strains did not show significant differences in their motility traits. The results demonstrate that M. phaseolina exudate contains chemical attractants that serve as signal for flagellar motility of P. fluorescens. Motile P. fluorescens strains thus may consume fungal exudate as nutrients, and thus spores could offer a niche for these bacteria in soil.     

Residual effects of natural bush,Cajanus cajan and Tephrosia candida on the productivity of an acid soil in southeastern Nigeria

An experiment was established in 1986 to examine the contribution of Tephrosia candida and Cajanus cajan shrubs to improving the productivity of an acid soil. The main treatments were N levels (0 and 60 kg ha^-1) with subplots of maize/natural bush, maize/Tephrosia candida, maize/Cajanus cajan, maize + cassava/natural bush, maize + cassava/Tephrosia candida, and maize + cassava/Cajanus cajan. In 1988, all plots were cleared and maize uniformly planted to study the residual effects of the treatments. No residual effects of N application were observed. Tephrosia candiada and Cajanus cajan increased surface soil organic carbon and total N levels over the natural bush. However, only Tephrosia candida plots produced improved maize grain and stover yield. Highly significant correlations were found between maize grain yield and earleaf N (r=0.73^**), grain N (r=0.51^**), and stover N (r=0.54^**) contents. These results suggest that Tephrosia candida increased N availability in the soil. Therefore, the shrub has potential for improving the productivity of acid soils under traditional systems, where N is limiting due to the absence of N₂-fixing legumes in the natural bush fallow.     

Chemotaxis of Rhizobium spp. to Plant Root Exudates

Rhizobium spp. show chemotaxis to plant root exudates. Both legumes and non-legume root exudates attract the different rhizobia studied. However, the bacteria show a differential response in that they are attracted to the root exudates of some plants and show no attraction toward others. An example of negative chemotaxis was also observed. The trefoil strain of Rhizobium shows chemotaxis which is qualitatively different from that observed in other bacteria in that simple sugars, di-and trisaccharides, dextrans, and amino acids do not attract this bacterium.     

Rhizosphere microorganism effects on soluble amino acids,sugars and organic acids in the root zone ofAgropyron cristatum,A. smithii andBouteloua gracilis

Three axenic and rhizosphere microorganism-inoculated shortgrass steppe plant species were evaluated for possible differences in residual organic carbon and nitrogen present as sugars, organic acids and amino acids. IntroducedAgropyron cristatum was compared toA. smithii andBouteloua gracilis, which are dominant species in the native shortgrass steppe. These plants, grown for 90 days in root growth chambers, showed differences in residual organic carbon and nitrogen per gram of root, and rhizosphere microbe presence resulted in additional changes in these compounds. The root biomass ofB. gracilis was significantly increased with microbes present. TheAgropyron species had significantly lower amino acid levels with microbes present, while under the same conditions, theB. gracilis showed significant decreases in residual sugars. Based on the amino acids, sugars and organic acids, the C/N ratio of the sterileA. cristatum was higher than forB. gracilis. Rhizosphere microbe presence did not result in changes in these C/N ratios. These results suggest thatA. cristatum, with microbes present, will have lower levels of amino acids present, whileB. gracilis, with a lower C/N ratio, will have sugars used to a greater extent by the rhizosphere microbes. This resulted in the higher levels of residual soluble organic C and N in the rhizosphere ofB. gracilis, in comparison with the introducedA. cristatum. These differences may be critical in influencing the course of nutrient accumulation and plant competition in short-grass steppe communities, and in understanding basic aspects of plant-rhizosphere microorganism interactions.