Growth regulators,Rhizobium and nodulation in peas

The cytokinin content of roots and nodules of pea and the culture supernatants from two strains of Rhizobium leguminosarum has been examined. Roots, nodules and wild-type Rhizobium culture medium contained very little cytokinin as indicated by bioassay. Chemical ionisation gas chromatography-mass spectrometric analysis of the isopentenyladenine content of the culture medium from the Rhizobium strains confirmed that the content of the wild-type was low (approx. 1 ng dm^-3) but that it was increased by the introduction of the Agrobacterium Ti plasmid into the Rhizobium strain.Abbreviations CI chemical ionisation - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - iPAde isopentenyladenine - MIM multiple ion monitoring     

Biological nitrogen fixation in non-leguminous field crops: Recent advances

There is strong evidence that non-leguminous field crops sometimes benefit from associations with diazotrophs. Significantly, the potential benefit from N₂ fixation is usually gained from spontaneous associations that can rarely be managed as part of agricultural practice. Particularly for dryland systems, these associations appear to be very unreliable as a means of raising the nitrogen status of plants. However, recent technical advances involving the induction of nodular structures on the roots of cereal crops, such as wheat and rice, offer the prospect that dependable symbioses with free-living diazotrophs, such as the azospirilla, or with rhizobia may eventually be achieved.     

Spontaneous nodules induce feedback suppression of nodulation in alfalfa

A small subpopulation of alfalfa (Medicago saliva L.) plants grown without fixed nitrogen can develop root nodules in the absence of Rhizobium. Cytological studies showed that these nodules were organized structures with no inter- or intracellular bacteria but with the histological characteristics of a normal indeterminate nodule. Few if any viable bacteria were recovered from the nodules after surface sterilization, and when the nodular content was used to inoculate alfalfa roots no nodulation was observed. These spontaneous nodules were formed mainly on the primary roots in the region susceptible to Rhizobium infection between 4 and 6 d after seed imbibition. Spontaneous nodules appeared as early as 10 d after germination and emerged at a rate comparable to normal nodules. The formation of spontaneous nodules on the primary root suppressed nodulation in lateral roots after inoculation with R. meliloti RCR2011. Excision of spontaneous nodules at inoculation eliminated the suppressive response. Our results indicate that the presence of Rhizobium is not required for nodule organogenesis and the elicitation of feedback regulation of nodule formation in alfalfa.Abbreviation RT root tip This work was supported by an endowment to the Racheff Chair of Excellence of the University of Tennessee, and the Soybean Promotion Board, Haskinsville, Tenn., USA. We are indebted to Noel Gerahty for performing the acetylene-reduction assays, and Dr. E.T. Graham for allowing the use of microscope facilities.     

Influence ofRhizobium leguminosarum biovartrifolii host specific nodulation genes on the ontogeny of clover nodulation

Summary The infection of white clover seedlings byRhizobium strains with different host range properties was assessed using various microscopic techniques. Several wild-type andRhizobium leguminosarum biovarvicias hybrid strains containing definedR. l. bv.trifolii host range genes were used. The morphological changes in the root tissue of uninoculated and rhizobia inoculated white clovers were identified and compared. In particular, changes were observed in the induction of inner cortical cell division, alterations to nodule development and lateral root formation. The responses of the infected roots and the types of structures formed support the hypothesis that lateral roots and nodules may be physiologically homologous structures. To establish a normal pattern of nodulation on white clover roots, both sets of known host specific nodulation genes (operonsnod FERL andnod MNX) ofR. l. bv.trifolii were required. However, some nodule development occurred when only thenod FERL genes were present in the hybrid strain.     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Use of a promoter-specific probe to identify two loci from the Rhizobium meliloti nodulation regulon

The nodulation regulon of Rhizobium meliloti AK631 includes several operons (nodABC, hsnABC, hsnD, efn locus) which have in common a consensus promoter sequence called the nod box. A synthetic nod box probe was used to identify two additional nod boxes, n4 and n5, which were subcloned for study. By constructing lac fusions, we show that n4 and n5 sponsor induction of downstream regions as previously shown for n1-nodABC and n2-hsnABC. Using site-directed Tn5 mutagenesis, we find that the n5 locus plays a significant role in nodulation of alfalfa and sweetclover, whereas the n4 locus is important for alfalfa, but not for sweetclover. Hybridization data suggest that the n5 locus is conserved among Rhizobium species. In contrast, the n4 locus seems to be unique to Rhizobium meliloti strains, in agreement with the host-specific phenotype of n4 locus mutants. Thus, the use of a promoter probe allows us to identify nodulation genes which may be overlooked by standard methods such as random Tn5 mutagenesis.     

Assessment of nodulation of Mucuna pruriens by promiscuous indigenous rhizobia in the moist savanna zone of Nigeria

Leucaena leucocephala failed to nodulate effectively with promiscuous indigenous rhizobia with which Mucuna pruriens nodulated effectively. Mucuna pruriens was adequately established and well nodulated due to the presence of favourable climatic and edaphic factors which enhanced its establishment in the humid/moist savanna zone of Nigeria. The microsymbiont for M. pruriens seems to thrive more in an alkaline rhizosphere. Introduction of M. pruriens into the farming system in Nigeria may serve as a low input agricultural system, which is not only sustainable but also economically viable.     

The Rhizobium meliloti early nodulation genes (nodABC) are nitrogen-regulated: Isolation of a mutant strain with efficient nodulation capacity on alfalfa in the presence of ammonium

Summary The presence of combined nitrogen in the soil suppresses the formation of nitrogen-fixing root nodules by Rhizobium. We demonstrate that bacterial genes determining early nodulation functions (nodABC) as well as the regulatory gene nodD3 are under nitrogen (NH ₄ ⁺ ) control. Our results suggest that the gene product of nodD3 has a role in mediating the ammonia regulation of early nod genes. The general nitrogen regulatory (ntr) system as well as a chromosomal locus mutated in Rhizobium meliloti were also found to be involved in the regulation of nod gene expression. A R. meliloti mutant with altered sensitivity to ammonia regulation was isolated, capable of more efficient nodulation of alfalfa than the wild-type strain in the presence of 2 mM ammonium sulfate.     

Effects of high temperature on nodulation and nitrogen fixation by Phaseolus vulgaris L.

Screening of Rhizobium leguminosarum bv. phaseoli strains showed some that were able to nodulate common beans (Phaseolus vulgaris L.) at high temperatures (35 and 38°C/8 h/day). The nodulation ability was not related to the capability to grow or produce melanin-like pigment in culture media at high temperatures. However, nodules formed at high temperatures were ineffective and plants did not accumulate N in shoots. Two thermal shocks of 40°C/8 h/day at flowering time drastically decreased nitrogenase activity and nodule relative efficiency of plants otherwise grown at 28°C. Recovery of nitrogenase activity began only after seven days, when new nodules formed; total incorporation of N in tops did not recover for 2 weeks. Non-inoculated beans receiving mineral N were not affected by the thermal shock, and when growing continuously at 35 or 38°C had total N accumulated in shoots reduced by only 18%.     

Plant responses to nodulation factors

The focus of research on signalling in Rhizobium-legume interactions has moved from understanding the structure and synthesis of rhizobially made Nod factors, towards an analysis of how they function in plants. Nod-factor-induced changes in ion fluxes across membranes, followed by establishment of an oscillation of intracellular Ca(2+) concentration, point to the involvement of a receptor-mediated signal transduction pathway. Progress towards the identification of components in this pathway is being made by identifying Nod-factor binding proteins, isolating plant mutants that are defective in signalling and analysing plant responses to Nod factors.     

Arbuscular mycorrhizal colonization and nodulation improve flooding tolerance in <Emphasis Type="Italic">Pterocarpus officinalis</Emphasis> Jacq. seedlings

Pterocarpus officinalis (Jacq.) seedlings inoculated with the arbuscular mycorrhizal fungus, Glomus intraradices, and the strain of Bradyrhizobium sp. (UAG 11A) were grown under stem-flooded or nonflooded conditions for 13 weeks after 4 weeks of nonflooded pretreatment under greenhouse conditions. Flooding of P. officinalis seedlings induced several morphological and physiological adaptive mechanisms, including formation of hypertrophied lenticels and aerenchyma tissue and production of adventitious roots on submerged portions of the stem. Flooding also resulted in an increase in collar diameter and leaf, stem, root, and total dry weights, regardless of inoculation. Under flooding, arbuscular mycorrhizas were well developed on root systems and adventitious roots compared with inoculated root systems under nonflooding condition. Arbuscular mycorrhizas made noteworthy contributions to the flood tolerance of P. officinalis seedlings by improving plant growth and P acquisition in leaves. We report in this study the novel occurrence of nodules connected vascularly to the stem and nodule and arbuscular mycorrhizas on adventitious roots of P. officinalis seedlings. Root nodules appeared more efficient fixing N₂ than stem nodules were. Beneficial effect of nodulation in terms of total dry weight and N acquisition in leaves was particularly noted in seedlings growing under flooding conditions. There was no additive effect of arbuscular mycorrhizas and nodulation on plant growth and nutrition in either flooding treatment. The results suggest that the development of adventitious roots, aerenchyma tissue, and hypertrophied lenticels may play a major role in flooded tolerance of P. officinalis symbiosis by increasing oxygen diffusion to the submerged part of the stem and root zone, and therefore contribute to plant growth and nutrition.     

Effects of ethylene and inhibitors of ethylene synthesis and action on nodulation in common bean (Phaseolus vulgaris L.)

The ethylene releasing compound, 2-chloroethylphosphonic acid (ethephon) inhibited nodule development in common bean (Phaseolus vulgaris L.) plants. In contrast, inhibitors of ethylene synthesis or its physiological activity enhanced nodulation. In a co-culture of bean seeds and rhizobia, ethephon inhibited rhizobial growth while inhibitors of ethylene synthesis or action did not influence the growth and proliferation of rhizobia. These data emphasize the role of ethylene as a regulator of nodulation in determinate nodulators and indicate that the ethylene signaling pathway involved in the nodulation process is not limited to the plant host but also involves the bacterial symbiont.     

Plant growth-promoting rhizobacteria for improving nodulation and nitrogen fixation in the common bean (Phaseolus vulgaris L.)

A greenhouse experiment was performed to evaluate the effects of plant growth-promoting rhizobacteria (PGPR) on nodulation, biological nitrogen fixation (BNF) and growth of the common bean (Phaseolus vulgaris L. cv. Tenderlake). Single and dual inoculation treatments of bean with Rhizobium and/or PGPR were administered to detect possible changes in the levels of and interactions between the phytohormones IAA and cytokinin. Bean plants cv. Tenderlake were grown in pots containing Fluvic Neosol eutrophic (pH 6.5). Fourteen kilogram aliquots of soil contained in 15-l pots were autoclaved. Bean seeds were surface sterilized and inoculated with Rhizobium tropici (CIAT 899-standard strain) alone and in combination with one of the PGPR strains: Bacillus endophyticus (DSM 13796), B. pumilus (DSM 27), B. subtilis (DSM 704), Paenibacillus lautus (DSM 13411), P. macerans (DSM 24), P. polymyxa (DSM 36), P. polymyxa (Loutit L.) or Bacillus sp.(65E180). The experimental design was randomized block design with three replications. Beans co-inoculated with Rhizobium tropici (CIAT899) and Paenibacillus polymyxa (DSM 36) had higher leghemoglobin concentrations, nitrogenase activity and N₂ fixation efficiency and thereby formed associations of greater symbiotic efficiency. Inoculation with Rhizobium and P. polymyxa strain Loutit (L) stimulated nodulation as well as nitrogen fixation. PGPR also stimulated specific-nodulation (number of nodules per gram of root dry weight) increases that translated into higher levels of accumulated nitrogen. The activities of phytohormones depended on their content and interactions with Rhizobium tropici and Paenibacillus and/or Bacillus (PGPR) strains which affect the cytokinin in content in the common bean.     

Effect of Rhizobium inoculation methodologies on nodulation and growth of Leucaena leucocephala

The aim of this study was to evaluate the effect of five methods of Rhizobium inoculum application on nodulation and nitrogen fixation in Leucaena leucocephala seedlings cultivated for 6 months in the greenhouse. Plants inoculated with alginate beads were significantly more developed and more nodulated than plants inoculated with the other methodologies used.     

Coculture of Pachyrhizus erosus (L.) hairy roots with Rhizobium spp.

Summary We have established an in vitro system for the induction and study of nodulation in Pachyrhizus erosus (jicama) via a hairy root-Rhizobium coculture. In vitro-grown P. erosus plantlets were infected with Agrobacterium rhizogenes (ATCC No. 15834) and two hairy root lines were established. Hairy roots were grown in a split-plate system in which compartment I (CI) contained MS medium with nitrogen and different sucrose levels (0–6%), while CII held MS medium without nitrogen and sucrose. Nodule-like structures developed in transformed roots grown in CI with 2–3% surcose, inoculated with Rhizobium sp. and transferred to CII. Nodule-like structures that developed from hairy roots lacked the rigid protective cover observed in nodules from plants grown in soil. Western blot analysis of nodules from hairy roots and untransformed roots (of greenhouse-grown jicama) showed expression of glutamine synthetase leghemoglobin and nodulins. Leghemoglobin was expressed at low levels in hairy root nodules.     

nodT,a positively-acting cultivar specificity determinant controlling nodulation of Trifolium subterraneum by Rhizobium leguminosarum biovar trifolii

Rhizobium leguminosarum biovar trifolii strain TA1 nodulates a range of Trifolium plants including red, white and subterranean clovers. Nitrogen-fixing nodules are promptly initiated on the tap roots of these plants at the site of inoculation. In contrast to these associations, strain TA1 has a Nod^- phenotype on a particular cultivar of subterranean clover called Woogenellup (A.H. Gibson, Aust J Agric Sci 19: (1968) 907–918) where it induces rare, poorly developed, slow-to-appear and ineffective lateral root nodules. By comparing the nodulation gene region of strain TA1 with that of another R. leguminosarum bv. trifolii strain ANU843, which is capable of efficiently nodulating cv. Woogenellup, we have shown that the nodT gene (B.P. Surin et al., Mol Microbiol 4: (1990) 245–252) is essential for nodulation on cv. Woogenellup. The nodT gene is naturally absent in strain TA1. A cosmid clone spanning the entire nodulation gene region of strain TA1 was capable of conferring nodulation ability to R.l. bv. trifolii strains deleted for nodulation genes, but only on cultivars of subterranean clovers nodulated by strain TA1. This shows that cultivar recognition events are, in part, determined by genes in the nodulation region of strain TA1. Complementation studies also indicated that strain TA1 contains negatively-acting genes located on the Sym plasmid and elsewhere, which specifically block nodulation of cv. Woogenellup.