Root Nodules Of Clover Research Articles

Extracellular Synthesis and Characterization of Silver Nanoparticles from Alkaliphilic Pseudomonas sp.

Bio-nanotechnology offers eco-friendly processes for the synthesis of stable nanoparticles (NPs). We hypothesized that microorganisms isolated from the root nodules of leguminous plants would biosynthesize silver (Ag) bio-nanoparticles. Clover root nodules enriched with nutrient broth (NB) produced four distinct colonies on NA plates. Microbial colonies were purified by repeated streaking and designated as SS6, SS7, SS8, and SS9 for identification using 16S rRNA sequencing. Four species of Pseudomonas were identified with a similarity score of over 99% using the EZ Taxon search engine, and tested for extracellular biosynthesis of AgNPs. Microorganism Pseudomonas taiwanensis-SS8 with alkaliphilic growth characteristics reduced the AgNO3 solution into AgNPs in the shortest time period. AgNPs were characterized using UV-Vis spectrophotometry and electron and transmission electron microscopy. A number of physical (i.e., temperature and time) and chemical (i.e., pH and growth media) parameters were optimized. An efficient polydispersal biosynthesis of AgNPs at pH 8-9 after 48 hrs in NB growth medium was observed. In addition, the AgNPs showed antimicrobial properties against 16 commonly occurring pathogenic microorganisms.

Biochar application reduces nodulation but increases nitrogenase activity in clover

Biochar is produced from the pyrolysis of organic materials, and when buried in soil can act as a long term soil carbon (C) store. Evidence suggests that biochar can also increase crop yields, reduce nutrient leaching and increase biological nitrogen fixation in leguminous plants. However, the potential for increasing biological N2 fixation in agroecosystems is poorly understood, with inconsistent reports of root nodulation following biochar application. Therefore, the aim of this study was to determine the effect of biochar application rate and time since application on nodulation and nitrogenase activity in nodules of clover grown in a temperate agricultural soil. We used replicated field plots with three biochar application rates (0, 25 and 50 t ha−1). Three years after biochar amendment, the plots were further split and fresh biochar added at two different rates (25 and 50 t ha−1) resulting in double-loaded reapplications of 25 + 25 and 50 + 50 t ha−1. Three years after biochar application, there was no significant difference in the total number of root nodules between biochar-amended and unamended soil, regardless of the application rate. However, despite clover root nodules being of a similar number and size the level of nitrogenase activity of individual nodules in biochar-amended soil was significantly higher than in unamended soil. Reapplication of biochar resulted in decreased nodulation, although the rate of nitrogenase activity per nodule remained unaffected. In the short term, biochar influences root nodule number and localised N2 fixation per nodule; however, total nitrogenase activity for the whole root system remained unaffected by the application rate of biochar or time since its application. These results emphasise the importance of long-term field studies, with a variety of applications rates for determining the influence of biochar applications on N2-fixing organisms and in providing data that can meaningfully inform agronomic management decisions and climate change mitigation strategies.

Genetic diversity of Romanian Rhizobium leguminosarum biovar.trifolii strains isolated from root nodules of clover grown in heavy metal polluted soil

This study was designed to determine the state of polluted soils in the main landfills of the Community of Madrid (central Spain), as part of a continuous assessment of the impacts of urban solid waste (USW) landfills that were capped with a layer of soil 20 years ago. Our analysis of this problem has been highly conditioned by the constant re-use of many of the USW landfills, since they have never been the target of any specific restoration plan. Our periodical analysis of cover soils and soils from discharge areas of the landfills indicates soil pollution has worsened over the years. Here, we examined heavy metal, salts, and organic compounds in soil and surface water samples taken from 15 landfills in the Madrid region. Impacts of the landfill soil covers on nematode and plant diversity were also evaluated. These analyses continue to reveal the presence of heavy metals (Zn, Cu, Cr, Ni, Pb, Cd) in soils, and salts (sulphates, chlorides and nitrates) in soils and surface waters. In addition, non-agricultural organic compounds, mainly aromatic and aliphatic hydrocarbons, often appeared in very high concentrations, and high levels of insecticides such as gamma-HCH (lindane) were also detected in soils. Around 50% of the water samples collected showed chemical demand of oxygen (CDO) values in excess of 150 mg/l. Traces of phenolic compounds were detected in some landfills, some of which exhibited high levels of 2-chlorophenol and pentachlorophenol. All these factors are conditioning both the revegetation of the landfill systems and the remediation of their slopes and terrestrial ecosystems arising in their discharge areas.This work updates the current situation and discusses risks for the health of the ecosystems, humans, domestic animals and wildlife living close to these landfills.

New Rhizobium leguminosarum bv. trifolii isolates: collection, identification and screening of efficiency in symbiosis with clover

Suitable clover stands (field and meadow) were chosen to isolate new R. leguminosarum bv. trifolii strains. Together 49 strains were isolated, labelled and maintained. Ten primers were used for identification and amplification products were detected by agarose electrophoresis. Amplification products of individual primers did not make possible to distinguish all isolates; a combination of at least two primers was necessary. No differences in genetic variability level between field and meadow isolates were found. Hydroponic perlite experiments were used for the screening of rhizobial isolates and the tetraploid red clover variety Amos was used as a host plant. Shoots were weighed and the root volume was determined at the onset of anthesis. Total nitrogenase activity (TNA) of the symbiotic system was measured and the root dry weight was determined. High differences among individual isolates were found in all the characteristics. Statistically significant were differences among individual isolates for TNA, growth characteristics did not differ significantly. Strong positive relationships between fresh forage, dry forage, root volume and root dry weight were determined (r = 0.69–0.98; p > 0.05). Nine isolates were selected as effective, based on the mean values of all isolates screened. Selected isolates achieved the above average values in most measured characteristics. Out of the nine isolates, two were isolated from root nodules of clover cultivated in field stands, seven strains were isolated from clover grown in natural meadows. Additional study of isolates in soil condition will follow.

Immunocytochemistry of Rhamnogalacturonan II in Cell Walls of Higher Plants

A polyclonal antibody against a borate-RG-II complex is raised in rabbits. The antibody recognized RG-II exclusively in cell wall polysaccharides. Immunocytochemical studies demonstrated that the epitope is ubiquitous in cell walls of all the cells in radish and rice roots, cultured tobacco cells, red clover root nodules, and lily growing pollen tubes. The label was denser in proximal to plasma membrane, and not detected in middle lamella, suggesting that borate may cross-link newly secreted pectic polysaccharides at the membrane-cell wall interface.

Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth

The aim of this study was to identifiy the endophytic bacteria recovered from the foliage, tap roots and nodules of red clover plants (Trifolium pratense L.); and to assess the effects of the nodule bacteria, alone and in combination with Rhizobium spp., on the growth and development of red clover seedlings. Thirty-one bacteria species from 14 different genera were recovered from within the foliage, roots and nodules of red clover plants cv. AC Charlie. Genera diversity and species number were greatest in foliage tissues. Pantoea agglomerans (59.6%) was the most frequent species recovered in foliage tissues, Agrobacterium rhizogenes A in the tap root (49.2%) and Rhizobium leguminosarum BV phaseoli and R. loti B in the nodules (27.2% each). Recovery of Rhizobium species was not restricted to the nodules, and species of this genus were systemic throughout the plant. Clover root nodules were host to 12 bacteria species other than rhizobia, of which 8 were specific to this tissue. Using non-selective media, R. leguminosarum BV trifolii constituted only 8.8% of all the root nodule bacteria recovered. In root bacterization experiments, species of nodule bacteria promoted growth of red clover more often when applied in combination with R. leguminosarum BV trifolii than when applied singly. However, Bacillus megaterium, Bordetella avium and Curtobacterium luteum consistently promoted growth either individually or in combination with R. leguminosarum BV trifolii. Nodulation was promoted when R. leguminosarum BV trifolii was coinoculated with Bacillus insolitus, B. brevis or A. rhizogenes A. Single isolate applications of Rhizobium species to roots always led to the depression of clover growth, but mixtures of R. leguminosarum BV trifolii and R. leguminosarum BV phaseoli resulted in growth promotion. The latter is considered further evidence of the beneficial allelopathic side effect of strain competition for the same ecological niche.

Differentiation of Rhizobium strains using the polymerase chain reaction with random and directed primers

Total genomic DNA isolated from Rhizobium spp was analysed by the polymerase chain reaction (PCR) in conjunction with either arbitrary oligonucleotide primers or a 20-base oligonucleotide primer corresponding to a conserved nif gene promoter region. The amplified fragment length polymorphisms (amplification profiles) generated by the 2 arbitrary primers (RPO4 and RPO5), effectively differentiated a diverse collection of Rhizobium meliloti, R. leguminosarum bv. trifolii and R. l. bv. viciae strains. The nif-directed primer (RPO1) was also highly discriminatory on Rhizobium DNA and generated unique amplification profiles for each strain. For selected strains, reproducible amplification profiles were obtained using a variety of DNA sources, and these were achievable over a 125-fold range in template concentration. Amplification profiles generated by the primers RPO5 and RPO1 were shown to be temperature dependant, with the RPO1 primer capable of generating amplification profiles at annealing temperatures up to 65°C. Reproducible amplification profiles were generated from either purified total genomic DNA or from template DNA present in freeze-thaw bacterial cell lysates. Moreover, the RPO1 primer was used to positively identify specific R. l. bv. trifolii strains directly in crude extracts prepared from squashed clover-root nodules. The significance of these results is discussed in relation to the development of strategies for using PCR-based methodologies in Rhizobium ecological studies.

Plasmid Profiles of Rhizobia Used in Inoculants and Isolated from Clover Fields

AbstractThe persistence of inoculant strains of Rhizobium is a factor determining the need for inoculation of legumes. Four pastures in east Texas, with known histories of arrowleaf [Trifolium vesiculosum Savi] and crimson [T. incarnatum L.] clover planting and inoculation with R. leguminosarum bv. trifolii, were used to investigate the prevalence of inoculant strains in clover root nodules. Three pastures had mixed stands of the two clover species, and the fourth had only arrowleaf clover. Using plasmid profiles as a means of identifying rhizobial strains, we analyzed 50 rhizobial isolates made from root nodules of each clover species in each of the pastures. Sixty‐six plasmid profile types were obtained from the 350 isolates. For the first three sites, only 2 of 300 isolates had plasmid profiles matching that of an inoculant strain, and 5 others had profiles similar but not identical to that of another inoculant strain. More than one‐half of the plasmid profile types corresponded to variations of a single type, distinctly different from that of any inoculant strain. At the site where only arrowleaf clover had been grown, 70% of the isolates had a plasmid profile identical to that of an inoculant strain not used at the site. The instability of the plasmid profile of the latter, when grown in vitro, and the similarity of its plasmid and total soluble protein profiles to those of the inoculant strains used at this site, indicate that the prevalent type probably originated from one of them. The inoculant strains failed to persist as such in the field, regardless of the frequency of inoculation.

Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness

Genes involved in nodulation competitiveness (tfx) were inserted by marker exchange into the genome of the effective strain Rhizobium leguminosarum bv. trifolii TA1. Isogenic strains of TA1 were constructed which differed only in their ability to produce trifolitoxin, an antirhizobial peptide. Trifolitoxin production by the ineffective strain R. leguminosarum bv. trifolii T24 limited nodulation of clover roots by trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii. The trifolitoxin-producing exconjugant TA1::10-15 was very competitive for nodulation on clover roots when coinoculated with a trifolitoxin-sensitive reference strain. The nonproducing exconjugant TA1::12-10 was not competitive for nodule occupancy when coinoculated with the reference strain. Tetracycline sensitivity and Southern analysis confirmed the loss of vector DNA in the exconjugants. Trifolitoxin production by TA1::10-15 was stable in the absence of selection pressure. Transfer of tfx to TA1 did not affect nodule number or nitrogenase activity. These experiments represent the first stable genetic transfer of genes involved in nodulation competitiveness to a symbiotically effective Rhizobium strain.

Observations on the diet of the Southern Brown Bandicoot, lsoodon obesulus (Marsupialia: Peramelidae), in southern Tasmania.

Analysis of 48 faecal samples of lsoodon obesulus, collected over three seasons from Huonville in southern Tasmania, revealed prey representing 36 plant and invertebrate taxa. I. obesulus appears to be a qualitatively opportunistic omnivore feeding on a wide range of invertebrate and plant material. Quantitatively, in terms of type of prey, a large number of disparities exist between arthropod prey items in the diet and in pitfall traps and soil samples. Principal invertebrate items comprised Formicidae, Scarabaeidae larvae, Staphylinidae beetles, Calliphoridae larvae and Araneae. Major plant items were grasses, seeds, clover root nodules, Gasteromycete and Zygomycete fungi. The diet appeared to reflect seasonally and locall y abundant food items.

Effect of a bacteriophage on the colonisation and nodulation of clover roots by a strain of Rhizobium trifolii.

The presence of a virulent bacteriophage in the root zone of clover growing in seedling agar under controlled environments (14--17 and 19--23 degrees C) produced changes in the persistence and symbiotic effectiveness of a susceptible strain of Rhizobium trifolii. The phage reduced the rhizoplane population of rhizobia and led to the appearance of variant substrains which were less susceptible to the bacteriophage and mostly ineffective in symbiotic nitrogen fixation. Some were also changed in colonial morphology and nutritional requirements. At the higher temperature, the frequency of bacterial variants increased and the number of nodules due to the parent strain decreased. A large initial population of bacteriophage was able to reduce, but generally did not completely suppress, nodulation.

Effect of a bacteriophage on colonisation and nodulation of clover roots by paired strains of Rhizobium trifolii.

The effect of a virulent bacteriophage on the competitive behaviour of paired strains of Rhizobium trifolii in the root zone of clover plants was examined. The presence of bacteriophage reduced the population density of susceptible strains on the root surface and, in nodulation, favoured resistant or even partially resistant strains which were otherwise less able to form nodules. These effects occurred with different growth temperatures, host species, and ratios of strains in the inoculum.

Viability of rhizobium trifolii bacteroids from clover root nodules

Rhizobium trifolii from protoplasts isolated from actively nitrogen-fixing nodules of white and subterranean clover show a colony forming efficiency of up to 90%. This indicates that these bacteria do not lose their viability when in the differentiated bacteroid state.

Pea root nodules containing more than one Rhizobium species

PARTICULAR species of legume are nodulated only by appropriate species of Rhizobium. Thus R. leguminosarum, R. phaseoli and R. trifolii nodulate peas, French beans and clover, respectively. The basis of this specificity between the two partners of the symbiosis is not understood. Complete specificity is not always found, however. Although R. trifolii does not normally nodulate peas, induced mutants of this species able to nodulate this host have been described1 and some strains of Rhizobium isolated from clover nodules nodulated both clover and peas2. Selected clover lines have been nodulated by R. leguminosarum3. It has been reported that a non-rhizobial contaminant can occur in soybean nodules4 and that the presence of R. trifolii may allow R. meliloti to occupy clover root nodules (B. O. Gillberg, personal communication). We show here that the presence of R. leguminosarum can allow R. trifolii and R. phaseoli to enter pea nodules but that R. trifolii cannot fix nitrogen in the nodules of this ‘foreign’ host.

Virus-like particles associated with the mycoplasmas of clover phyllody in the plant and in the insect vector.

SUMMARY: In a few phloem cells in root nodules of clover (Trifolium repens L.) and salivary glands of Euscelis lineolatus Brullé, which were naturally or experimentally infected by the mycoplasmas of clover phyllody, rod-shaped virus-like particles were found which had at least one rounded end. These particles had a diameter of 27 ± 3 nm, a dense core of diameter 16 ± 2nm and a clear axis. Their mean length was between 50 and 90 nm. Even when they were free in the phloem sap of the plant or in the haemolymph of the insect they were in the vicinity of some mycoplasmas. Sometimes they were crowded in a monolayer inside the cytoplasm of the plant cell. They were also found in the form of a rosette around some mycoplasmas which seemed degenerated. The occurrence, size, shape, internal structure and position of these virus-like particles is illustrated; their nature and pathogenicity in comparison with Mycoplasmatales Virus laidlawii 1 and the virus-like particles sometimes associated with plant pathogenic mycoplasmas is discussed.

Effects of copper deficiency on root nodules of subterranean clover

Effects of copper deficiency on the development of subterranean clover root nodules were studied. These included a depression of the bacteroid content, an accumulation of starch in the invaded cells and a reduction in the rate of development of the nodule apex. A similar pattern of increasing severity of metabolic effects in the youngest tissues was also found in leaves, but at a later stage of plant development than in nodules. At low copper supply the copper content of tissues and the activity of known copper-containing enzymes was depressed.

The origin of the membrane envelope surrounding the bacteria and bacteroids and the presence of glycogen in clover root nodules

Thin sections of clover nodules were examined by electron microscopy. The emergence of the bacteria from the infection thread was found to occur by a process of phagocytosis. No evidence was found, using glutar-aldehyde-osmium as a fixative, that there is any connection between the membrane envelope and the endoplasmic reticulum.

The structure of infection threads, bacteria and bacteroids in pea and clover root nodules

The bacteria and infection threads of the pea root nodule were examined by light and electron microscopy. The bacteria in the infection thread are enclosed in a microcapsule. This capsule disappears when the bacterium is released into the host cytoplasm. The membrane envelope which surrounds the bacteria in the host cell is shown to be derived from the plant cell membrane. The infection of the host cell is by means of a process akin to pinocytosis and the bacteria are confined to vacuoles in the host cytoplasm. As each membrane envelope contains only one bacterium, the envelopes must divide with the bacteria. The bacteria increase 40fold in volume from the infection thread to the stage of the mature bacteroid. The mature infected host cell contains few organelles. The mitochondria become confined to the periphery of the cell. Differences of membrane structure in gram negative bacteria found by other workers have been attributed to fixation artifacts.