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.     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

Effect of nitrate supply on the in-vivo synthesis and distribution of trifollin A,a Rhizobium trifolii-binding lectin,in Trifolium repens seedlings

In-vivo synthesis of the white-clover lectin, trifoliin A, was examined by the incorporation of labeled amino acids into protein during heterotrophic growth of intact Trifolium repens L. seedlings. Lectin synthesis was quantified by measuring the level of labeled protein immunoprecipitated from root exudate, from the hapten (2-deoxyglucose) eluate of the roots, and from root and shoot homogenates. The presence of labeled trifoliin A was confirmed by non-denaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by fluorography and comparison with trifoliin A standards. In-vivo-labeled trifoliin A was detected in seedling root homogenate 2 h after the addition of labeled amino acids and on the root surface by 8 h. Incorporation of labeled amino acids into protein and trifoliin A was greatest with 2-d-old seedlings and was greater when the plants were grown continuously in the dark than when they were exposed to 14 h light daily. Significantly more labeled lectin accumulated on the root surface of seedlings grown with 1.5 mM KNO₃ than of seedlings grown either without N or with 15.0 mM KNO₃. The labeled lectin from the root surface in all nitrate treatments and from the rootexudate samples of seedlings grown N-free and with 1.5 mM KNO₃ was fully able to bind to Rhizobium trifolii. In contrast, only 2% of the immunoprecipitable protein found in the root exudate of seedlings grown with 15.0 mM KNO₃ was able to bind to the bacteria. Thus, excess nitrate does not repress the synthesis of trifoliin A in the root, but does affect the distribution and activity of this newly synthesized lectin in a way which reduces its ability to interact with R. trifolii. By using Western blot analysis, much more total trifoliin A is detected in the homogenates of shoots than roots. However, greater than 80% of the total labeled protein and 85–90% of the total labeled lectin were found in the root homogenates of 2-d-old dark-grown seedlings incubated for 5 h with labeled amino acids. In addition, Western blot analysis indicated that the shoot homogenate contained smaller-molecular-weight peptides which reacted with the specific anti-trifoliin A antibody. These studies indicate that stored trifoliin A in the seed is degraded in the shoots during seedling development, while newly synthesized trifoliin A in the roots is excreted to the root surface and external environment.Abbreviations IgG immunoglobulin G - LPS lipopolysaccharide - PBS 10 mM potassium-phosphate buffer, pH 7.0, containing 0.8% NaCl - PBS-T 20 mM phosphate-buffered saline, pH 7.4, containing 0.05% Tween 20 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The response of white clover to different strains ofRhizobium trifolii in hill land reseeding: A second trial

Summary S. 184 white clover was surface seeded into natural molinia pasture on wet stagnogley soil containing no indigenousRhizobium trifolii. Seedlings were ‘spray inoculated’ after emergence with each of three strains ofR. trifolii. The best of these treatments produced an eight fold improvement in dry matter in the seeding year, followed by a 28% improvement in the following year. The results confirm the potential benefits which may be achieved by inoculating clover with suitable strains of rhizobia. The data are compared with a previously reported trial on an adjacent site where benefits were much greater in the first year. The difference is attributed to the overall advantages conferred in the present trial by much higher seedling populations and less severe competition from native species in the establishment phase.     

Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10⁶ bacteria·ml^-1) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.Abbreviations FITC fluorescein isothiocyanate - TRIC tetramethylrhodamine isothiocyanate     

Role of birds in dissemination of the thread blight disease caused byCylindrobasidium argenteum

The thread blight disease of broad leaved trees caused byCylindrobasidium argenteum occurred in laurel forests over 20 yr old after clear cutting in Kyushu. Finding fruit bodies of the causal fungus was possible but very difficult in the forests. In inoculation tests using spores and infected branches harvested in the forests, we succeeded in transmission only with branches. In the forests, the disease is transmitted almost entirely by contact with infected branches. During 1989 to 1992 we found four brown-eared bulbul nests containing branches infected with the disease. In one nest, mycelia of the fungus had been transmitted to the host tree. The occurrence of the disease in forests over 20-yr-old was considered to be due to the nesting of birds in trees which provided the necessary height and closed environment.     

The effect of sulfur supply on the root characteristics of subterranean clover and annual ryegrass

Summary The root characteristics and sulfur absorption of Trikkala sub-clover and Wimmera ryegrass were compared in a pot experiment in which plants were grown at two levels of sulfur supply (0 and 64 mgS/pot) for a period of 40 days after emergence.Ryegrass roots had a greater weight and root weight ratio, but a lower sulfur content than sub-clover roots at both levels of sulfur supply. Ryegrass roots were longer, had a greater length per unit weight, longer root hairs, and hence a larger volume and surface area than sub-clover roots. However, when the sulfur content in the whole plant was related to the root parameters, sub-clover absorbed a far greater amount of sulfur, irrespective of whether the content was expressed on the basis of the weight, length, volume or surface area of the roots.Sulfur application had relatively little effect on the morphology of roots of either species. However, whilst sulfur application increased the weight of both shoots and roots, it decreased the root weight ratio for sub-clover but not for ryegrass. Increasing sulfur supply reduced the total length of sub-clover roots but tended to increase the length of ryegrass roots. For both species, the length per unit weight of roots decreased, and the average root diameter increased, with increasing sulfur supply. Sulfur application decreased the volume and surface area of sub-clover roots, but increased these parameters in ryegrass roots.     

Persistence and recovery of introduced Rhizobium ten years after inoculation on Leucaena leucocephala grown on an Alfisol in southwestern Nigeria

Establishment of Leucaena leucocephala was poor at Ibadan (Transition forest-savanna zone) and Fashola (savanna zone, 70 km north of Ibadan) in southwestern Nigeria as a result of low soil fertility and the presence of only a few native rhizobia capable of nodulating it. Inoculation with L. leucocephala at these two locations in 1982 resulted in striking responses with Rhizobium strains IRc 1045 and IRc 1050 isolated from L. leucocephala grown in Nigeria. The persistence of inoculated effective Rhizobium strains after inoculation is desirable since it removes the need for reinoculation. Because of the perennial nature of L. leucocephala and its use in long-term alley farming experiments, we examined the persistence of inoculated rhizobial strains after inoculation, and their ability to sustain N₂-fixation and biomass production at Ibadan. In 1992, ten years after Rhizobium introduction, uninoculated, L. leucocephala fixed about 150 kg N ha^-1 yr^-1 or about 41% of total plant N compared to 180 kg N ha^-1 yr^-1 or 43% measured in 1982. Serological typing of the nodules using the Enzyme-Linked-Immunosorbent Assay (ELISA) and intrinsic resistance to the streptomycin test revealed that most of the nodules (96%) formed on L. leucocephala in 1992 were by Rhizobium strains IRc 1045 and IRc 1050, which were inoculated in 1982. Nodules were absent on uninoculated L. leucocephala grown on the adjacent field with no history of L. leucocephala cultivation. We conclude that the N₂ fixed by Rhizobium strains IRc 1045 and IRc 1050 persisted for many years in the absence of L. leucocephala and sustained effectively fixed N₂ which growth and yield of L. leucocephala after several years, thus encouraging a possible low-input alley farming system by smallholder farmers in Nigeria.     

Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance

Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.     

The ethylene-inhibitor aminoethoxyvinylglycine restores normal nodulation by Rhizobium leguminosarum biovar. viciae on Vicia sativa subsp. nigra by suppressing the ‘Thick and short roots’ phenotype

Nodulation of Vicia sativa subsp. nigra L. by Rhizobium bacteria is coupled to the development of thick and short roots (Tsr). This root phenotype as well as root-hair induction (Hai) and root-hair deformation (Had) are caused by a factor(s) produced by the bacteria in response to plant flavonoids. When very low inoculum concentrations (0.5–5 bacteria·ml^-1) were used, V. sativa plants did not develop the Tsr phenotype and became nodulated earlier than plants with Tsr roots. Furthermore, the nodules of these plants were located on the primary root in contrast to nodules on Tsr roots, which were all located at sites of lateral-root emergence. The average numbers of nodules per plant were not significantly different for these two types of nodulation. Root-growth inhibition and Hai, but not Had, could be mimicked by ethephon, and inhibited by aminoethoxyvinylglycine (AVG). Addition of AVG to co-cultures of Vicia sativa and the standard inoculum concentration of 5·10⁵ bacteria·ml^-1 suppressed the development of the Tsr phenotype and restored nodulation to the pattern that was observed with very low concentrations of bacteria (0.5–5 bacteria·ml^-1). The delay in nodulation on Tsr roots appeared to be caused by the fact that nodule meristems did not develop on the primary root, but only on the emerging laterals. The relationship between Tsr, Hai, Had, and nodulation is discussed.Abbreviations AVG aminoethoxyvinylglycine - cfu colonyforming units - Had root-hair deformation - Hai root-hair induction - NB naringenin-bacteria filtrate - Tsr Thick and short roots     

Molecular basis of the establishment and functioning of a N2-fixing root nodule

Compatible interactions between rhizobia and their leguminous host plant(s) culminate in the formation of a new plant organ, the root nodule. Within this structure, the bacteria reduce N₂ to NH₃ which is then assimilated by the plant. The formation of a N₂-fixing nodule requires a continuous process of two-way signalling and cellular recognition between the prokaryote and the plant. Such a process involves the sequential activation and/or repression of host plant- and bacteria-encoded genes. Finally, functioning of a legume-nodule necessitates not only the adaptation of plant and bacterial carbon, nitrogen and oxygen metabolism to an environment allowing N₂-fixation to occur, but also requires a tight co-ordination and integration of these plant and bacterial metabolic processes.     

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.     

A mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not of beans by R. phaseoli and is corrected by cloned DNA from Rhizobium or the phytopathogen Xanthomonas

Summary A Tn5-induced mutant strain of R. phaseoli which failed to synthesize exopolysaccharide (EPS) was isolated and was shown to induce normal nitrogen-fixing nodules on Phaseolus beans, the host of this Rhizobium species. The corresponding wild-type Rhizobium DNA was cloned in a wide host-range vector and by isolating Tn5 insertions in this cloned DNA, mutations in a gene termed pss (polysaccharide synthesis) were isolated. These were introduced by marker exchange into near-isogenic strains of R. leguminosarum and R. phaseoli which differed only in the identity of their symbiotic plasmids. Whereas the EPS-deficient mutant strain of R. phaseoli induced normal nitrogen-fixing nodules on Phaseolus beans, the same mutation prevented nodulation of peas by a strain of R. leguminosarum which normally nodulates this host. Further, it was found that DNA cloned from the plant pathogen Xanthomonas campestris pathover campestris could correct the defect in EPS synthesis in R. leguminosarum and R. phaseoli and also restored the ability to nodulate peas to the pss::Tn5 mutant strain of R. leguminosarum.     

Phosphorus deficiency in red clover promotes exudation of orobanchol,the signal for mycorrhizal symbionts and germination stimulant for root parasites

Plant derived sesquiterpene strigolactones, which have previously been characterized as germination stimulants for root parasitic plants, have recently been identified as the branching factors which induce hyphal branching morphogenesis, a critical step in host recognition by arbuscular mycorrhizal (AM) fungi. We show here that, in red clover plants (Trifolium pratense L.), which is known as a host for both AM fungi and the root holoparasitic plant Orobanche minor Sm., reduced supply of phosphorus (P) but not of other elements examined (N, K, Mg, Ca) in the culture medium significantly promotes the release of a strigolactone, orobanchol, by the roots of this plant. In red clover plants, the level of orobanchol exudation appeared to be regulated by P availability and was in good agreement with germination stimulation activity of the root exudates. This implies that under P deficiency, plant roots attract not only symbiotic fungi but also root parasitic plants through the release of strigolactones. This is the first report demonstrating that nutrient availability influences both symbiotic and parasitic interactions in the rhizosphere.     

Accumulation of gentisic acid as associated with systemic infections but not with the hypersensitive response in plant-pathogen interactions

In the present work we have studied the accumulation of gentisic acid (2,5-dihydroxybenzoic acid, a metabolic derivative of salicylic acid, SA) in the plant-pathogen systems, Cucumis sativus and Gynura aurantiaca, infected with either prunus necrotic ringspot virus (PNRSV) or the exocortis viroid (CEVd), respectively. Both pathogens produced systemic infections and accumulated large amounts of the intermediary signal molecule gentisic acid as ascertained by electrospray ionization mass spectrometry (ESI-MS) coupled on line with high performance liquid chromatography (HPLC). The compound was found mostly in a conjugated (β-glucoside) form. Gentisic acid has also been found to accumulate (although at lower levels) in cucumber inoculated with low doses of Pseudomonas syringae pv. tomato, producing a nonnecrotic reaction. In contrast, when cucumber was inoculated with high doses of this pathogen, a hypersensitive reaction occurred, but no gentisic-acid signal was induced. This is consistent with our results supporting the idea that gentisic-acid signaling may be restricted to nonnecrotizing reactions of the host plant (Bellés et al. in Mol Plant-Microbe Interact 12:227–235, 1999). In cucumber and Gynura plants, the activity of gentisic acid as inducing signal was different to that of SA, thus confirming the data found for tomato. Exogenously supplied gentisic acid was able to induce peroxidase activity in both Gynura and cucumber plants in a similar way as SA or pathogens. However, gentisic-acid treatments strongly induced polyphenol oxidase activity in cucumber, whereas pathogen infection or SA treatment resulted in a lower induction of this enzyme. Nevertheless, gentisic acid did not induce other defensive proteins which are induced by SA in these plants. This indicates that gentisic acid could act as an additional signal to SA for the activation of plant defenses in cucumber and Gynura plants.     

The brassinosteroid growth response in pea is not mediated by changes in gibberellin content

The objective of this study was to increase our understanding of the relationship between brassinosteroids (BRs) and gibberellins (GAs) by examining the effects of BR deficiency on the GA biosynthesis pathway in several tissue types of pea (Pisum sativum L.). It was suggested recently that, in Arabidopsis, BRs act as positive regulators of GA 20-oxidation, a key step in GA biosynthesis [Bouquin et al. (2001) Plant Physiol 127:450–458]. However, this may not be the case in pea as GA₂₀ levels were consistently higher in all shoot tissues of BR-deficient (lk and lkb) and BR-response (lka) mutants. The application of brassinolide (BL) to lkb plants reduced GA₂₀ levels, and metabolism studies revealed a reduced conversion of GA₁₉ to GA₂₀ in epi-BL-treated lkb plants. These results indicate that BRs actually negatively regulate GA₂₀ levels in pea. Although GA₂₀ levels are affected by BR levels, this does not result in consistent changes in the level of the bioactive GA, GA₁. Therefore, even though a clear interaction exists between endogenous BR levels and the level of GA₂₀, this interaction may not be biologically significant. In addition to the effect of BRs on GA levels, the effect of altered GA₁ levels on endogenous BR levels was examined. There was no significant difference in BR levels between the GA mutants and the wild type (wt), indicating that altered GA₁ levels have no effect on BR levels in pea. It appears that the BR growth response is not mediated by changes in bioactive GA levels, thus providing further evidence that BRs are important regulators of stem elongation.