The effect of acidity on the production of signal molecules by Medicago roots and their recognition by Sinorhizobium

The Medicago sativa-Sinorhizobium symbiosis is challenged by acidity, resulting in generally poor nodulation and production. Medicago murex, however, can nodulate and grow at low pH. The effect of low pH on signal exchange in the Sinorhizobium-Medicago symbiosis was studied to gain a greater understanding of the basis for poor nodulation of M. sativa compared to M. murex. Root exudates from M. sativa and M. murex grown in buffered nutrient solution at pH 4.5, 5.8 and 7.0, were collected to measure the expression of nodB induction in Sinorhizobium. A nodB-gusA fusion was constructed and inserted into Sinorhizobium medicae strains WSM419 (acid tolerant) and CC169 (acid sensitive). We identified greater induction by root exudates from both Medicago spp. collected at pH 4.5 than at pH 5.8 and 7.0, less induction by M. murex than M. sativa and less induction of WSM419 than CC169. The same major inducing compounds, 4′,7-dihydroxyflavanone (liquiritigenin), 4′,7-dihydroxyflavone, and 2′,4′,4-trihydroxychalcone (isoliquiritigenin), were identified in exudates of M. murex and M. sativa at all pH values, although in increasing amounts at lower pH. Poor nodulation of M. sativa relative to M. murex under acid conditions is not the consequence of decreased induction of Sinorhizobium nodB by chemical inducers present in the root exudates of both species at low pH.     

Influence of pH and calcium on the growth,polysaccharide production and symbiotic association of<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis> SEMIA 116 with alfalfa roots

The Sinorhizobium -legume interaction is sensitive to a number of environmental factors, soil acidity being one of the most important. In the typic Hapludoll soil of the central-southern region of Córdoba (Argentine) it was found that the nodulation of alfalfa ( Medicago sativaL.) roots was impaired with a reduction in shoot dry matter under conditions of soil acidity (pH 5.5) Our results showed that the addition of lime as dolomitic limestone at a concentration of 1 t ha ^-1to acid soil caused a significant increase in the nodulation of alfalfa roots inoculated with the strain Sinorhizobium meliloti SEMIA 116 (recommended inoculum for alfalfa) in the greenhouse experiments. The success of the lime treatment may be related not only to an increase in the pH values but also to an increase in the Ca concentration, improving the growth of S. meliloti and its nodulation ability under acidic conditions. In this study, we also demonstrated an increment in the bacterial growth rate as well as in the production of exopolysaccharides and lipopolysaccharides under low pH (5.5) and a high Ca concentration (5 mM) in the culture medium.     

Genetic diversity of rhizobia associated with alfalfa in Serbian soils

We have evaluated the genetic diversity and phylogeny of alfalfa rhizobia, originating from different types of soils in Serbia and their ability to establish an effective symbiosis with alfalfa (Medicago sativa L.). A collection of 65 strains isolated from root nodules of alfalfa were characterized by rep-PCR analysis, partial and complete 16S rDNA gene and recA gene sequencing, as well as atpD gene sequencing and DNA–DNA hybridizations. The results of the sequence analyses revealed that Sinorhizobium meliloti is the dominant species in alfalfa nodules. Only one strain was identified as Sinorhizobium medicae, two strains as Rhizobium tibeticum and one strain as Rhizobium sp. Despite the fact that the majority of strains were identified as S. meliloti, a high genetic diversity at strain level was detected. Almost all isolates shared the ability to nodulate and fix nitrogen with M. sativa, except 11 of them, which were incapable of fixing nitrogen with this species. About 50% of the isolates showed values of symbiotic effectiveness (SE) above 50%, while 10% of the strains were highly effective with SE values above 70%. Some of the strains which were highly effective in nitrogen fixation at the same time could intensively solubilize phosphates, offering a possibility for multipurpose inoculum development. This was the first genetic study of rhizobia isolated from this region and also the first report of natural presence of R. tibeticum in root nodules of M. sativa.     

The effect of rhizobiophages on<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis> -<Emphasis Type="Italic"> Medicago sativa</Emphasis> symbiosis

Using Tn5 mutagenesis of lysogenic Sinorhizobium meliloti strain L5–30 (L54), three neomycin-resistant transconjugants differing in phage-resistance profiles were isolated. Two of them increased the dry weight of plants and were capable of establishing root nodules, whereas the third one was ineffective. The bacterium-phage interaction did not have observable consequences in the Medicago sativa - S. meliloti symbiosis because it did not affect the number of nodules on M. sativa or plant dry weight.     

Diversity of rhizobia isolated from an agricultural soil in Argentina based on carbon utilization and effects of herbicides on growth

Seventy-six rhizobial isolates belonging to four different genera were obtained from the root nodules of several legumes (Vicia sativa, Vicia faba, Medicago sativa, Melilotus sp., Glycine max and Lotus corniculatus). The action of five commonly used herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate (GF), dicamba, atrazine and metsulfuron-methyl] on the growth of rhizobial strains was assessed. Subsequently, GF and 2,4-D were tested in a minimum broth as C and energy sources for 20 tolerant strains. The ability of these strains to metabolize different carbon sources was studied in order to detect further differences among them. Tolerance of the bacteria to agrochemicals varied; 2,4-D and GF in solid medium inhibited and diminished growth, respectively, in slow-growing rhizobial strains. Among slow-growing strains we detected Bradyrhizobium sp. SJ140 that grew well in broth + GF as the sole C and energy source. No strain was found which could use 2,4-D as sole C source. The 20 strains studied exhibited different patterns of C sources utilization. Cluster analysis revealed three groups, corresponding to four genera of rhizobia: Rhizobium (group I), Sinorhizobium (group II) and Mesorhizobium–Bradyrhizobium (group III). On the basis of the results obtained on responses to herbicides and C sources utilization by the isolates investigated, it was possible to differentiate them at the level of strains. These results evidenced a considerable diversity in rhizobial populations that had not been previously described for Argentinean soils, and suggested a physiological potential to use natural and xenobiotic C sources.     

PHOSPHORUS NUTRITION AND MYCORRHIZAL GROWTH RESPONSE OF BROADLEAF AND NARROWLEAF BIRDSFOOT TREFOILS

In Argentina narrowleaf trefoil (Lotus glaber) predominates over broadleaf trefoil (Lotus corniculatus). Researchers have suggested this predominance occurs because L. glaber grows better at low levels of P availability. I studied the effect of increasing phosphorus (P) nutrition and mycorrhizal growth response of Lotus corniculatus and Lotus glaber in a soil of low available P. L. corniculatus was more efficient in P utilization than L. glaber and produced larger yields of shoot tissue per unit of P. The critical P concentration, measured as a percentage of P in the shoot required to achieve 90% of maximum shoot yield, was 0.22 in L. corniculatus and 0.28 in L. glaber. The roots of both two species were heavily infected by arbuscular micorrhizae (AM), and both mycorrhizal and nonmycorrhizal plants responded strongly to added P in soil. This suggests that both species are at no advantage or disadvantage whether mycorrhizal or nonmicorrhizal. The fraction of roots colonized by AM fungi differed between species at low levels of added P but was similar at high levels of added P. The specific root length (cm g^?1) of mycorrhizal plants was greatest in L. corniculatus when P was insufficient to achieve maximum growth. The lower critical P concentration and the higher specific root length at low levels of P nutrition may be why L. corniculatus is superior to L. glaber in soils low in P. Therefore, the predominance of L. glaber compared with L. corniculatus in Argentinean soils is not due to ability of L. glaber to grow better in soil at low levels of P availability.     

Symbiotic effectiveness of Bradyrhizobium japonicum in acid soils can be predicted from their sensitivity to acid soil stress factors in acidic agar media

In acid soil, low pH, reduced availability of nutrients, and toxicity of Al and Mn limit plant growth and the survival and effectiveness of rhizobia. The symbiosis between legumes and rhizobia is particularly sensitive to acid soil stress. A pot experiment evaluated whether Bradyrhizobium japonicum strain growth on acidic agar media would predict ability to colonize the rhizosphere and form effective nodules in acidic soils. Three Indonesian strains of B. japonicum with similar effectiveness at neutral pH in sand culture but with different tolerance of acid soil stress factors in agar media, and an acid-tolerant commercial strain (CB1809) of comparable effectiveness, were tested in three acid soils using the Al tolerant soybean (Glycine max cv PI 416937). At 7 days after inoculation all strains had achieved large rhizosphere populations, but by day 14 the rhizosphere population of the acid-sensitive strain had decreased, while the more acid-tolerant strains increased. The acid-tolerant strains had significantly greater nodulation and symbiotic effectiveness than plants inoculated with the acid-sensitive strain. Laboratory prescreening of B. japonicum for acid, Al and Mn tolerance in acid media successfully identified strains which were symbiotically competent in low pH soils.     

Competition for nodule occupancy between introduced and native strains of Rhizobium leguminosarum biovar trifolii

The aim of this work was to evaluate the competitive ability between Rhizobium leguminosarum bv trifolii strain U204 used as commercial inoculants in Uruguay for Trifolium repens L. and Trifolium pratense L. and two native strains isolated from inoculated pastures of T. pratense. T126 is an efficient nitrogen fixer and a melanin producer strain; T70 is inefficient and a melanin non-producer strain; and U204 is very efficient in both hosts but is a melanin non-producer strain. Competitiveness between the strains was determined in experiments in pots and in growth pouches under controlled conditions. In the last experiment, we evaluated pH of plant nutrient solution and inoculum ratios. Plant dry weight was determined, and the identification of nodule bacteria was done using melanin production and DNA fingerprinting (GTG₅-PCR). The U204 symbiotic efficiency was not affected by the co-inoculation with the others two native strains. The T70 strain was a poor competitor when was co-inoculated with one of the effective strains in both experiments. Our results confirmed a “selective nodulation” because an effective symbiosis occurred preferentially over an ineffective one in Trifolium species. The native effective strain competed with U204 for nodule formation in both clovers species, but the nodule occupancy depended on the inoculum ratio. The pH of nutritive solution did not affect competition ability of the studied strains. It may be possible to isolate efficient, competitive, and genetically different native rhizobial strains to be used as inoculant strains for clover pastures in Uruguay. Both (GTG)₅-PCR and melanin production were useful methods to identify nodulating bacteria in competition studies.     

Cu-tolerant Sinorhizobium meliloti strain is beneficial for growth,Cu accumulation,and mineral uptake of alfalfa plants grown in Cu excess

In this work, we have compared the physiological responses of alfalfa plants inoculated with either Sinorhizobium meliloti strain S412 (Cu-tolerant) or S112 (Cu-sensitive) in the presence or absence of 0.5 mM of CuSO₄. The addition of copper (Cu) introduced a decrease of nodule number and their dry weight (DW) in both symbioses. The interaction established with the Cu-sensitive strain is more affected by Cu than that with the tolerant one. In fact, plants inoculated with the sensitive strain revealed a decrease of shoot and root DW, larger than that found in plants inoculated with the tolerant strain. However, under copper supply, Medicago sativa with the Cu-tolerant strain did not show any significant changes in both shoot and root biomass production. Under Cu excess, high levels of Cu were detected in different parts of the plant in the two symbioses and a high translocation of Cu to aerial parts was shown with the strain S412. Plants with S412 were able to accumulate large quantities of calcium (Ca) in their roots and nodules. While Ca content decreased drastically in shoot at 0.5 mM of Cu treatment. Moreover, nodulation with S412 allowed plants to maintain high levels of magnesium (Mg) in all tissues and high iron (Fe) levels in nodules. Results suggest that this symbiotic pair could be used in phytostabilization of Cu-contaminated soils.     

Inoculation with Phosphate Solubilizing Mesorhizobium Strains Improves the Performance of Chickpea (Cicer aritenium L.) Under Phosphorus Deficiency

The use of phosphate-solubilising bacteria as inoculants increases plant phosphorus (P) uptake and thus crop yield. Strains from the genus Mesorhizobium are among the most powerful phosphate solubilizing microorganisms. In order to study efficiency in P uptake and N₂ fixation in chickpea (Cicer aritenium), forty-two rhizobia strains natively from Tunisian soils were studied in symbiosis with the chickpea variety “Béja1” which is frequently cultivated in Tunisia. Plants were inoculated separately with these strains under controlled conditions in perlite under two sources of P i.e. soluble (KH₂PO₄) and insoluble P (Ca₂HPO₄). At flowering stage, growth, nodulation, P uptake and N₂ fixation were assessed in all symbiotic combinations. The results showed that the S27 strain efficiently mobilized P into plants, observed as a significant increase of plant P content when insoluble P (Ca₂HPO₄) was supplied to the soil. This was associated with a significant increase in plant biomass, nodule number and N content under insoluble P conditions. Additionally, inoculation with the Mesorhizobium strain S27 significantly increased the root acid phosphatase activity under insoluble P. This study also shows significant correlations found between plant P content and acid phosphatase activity under low P conditions which may highlight the contribution of acid phosphatases in increasing P use efficiency. A field experiment also showed that most of the chickpea analyzed parameters were improved when plants inoculated with two selected rhizobia strains (S26 and S27) and supplied with P2O5. Overall, these findings postulate that rhizobial inoculation should not only be based on the effectiveness of strains regarding N fixation, but also to other traits such as P solubilisation potential.     

Überleben inokulierter Rhizosphärenbakterien und deren Einfluß auf native Bakterienpopulationen im Wurzelraum von Luzerne

Survival of inoculated rhizosphere bacteria and their influence on native bacterial populations in the rhizosphere of alfalfa The survival of inoculated bacteria and their influence on native bacterial populations in the rhizosphere of alfalfa were investigated in a greenhouse experiment. The plant growth promoting strains Rhizobium meliloti me18 and Pseudomonas fluorescens PsIA12 were reisolated from the rhizosphere about 7 weeks after single and mixed strain inoculation. They did not induce lasting changes in the diversity of the native bacterial communities of the rhizosphere. Only within the first week after inoculation was an increase in total bacterial abundance observed. In general, the diversity of bacterial communities increased with plant age and with proximity to the root tip.     

A microcosm study on the influence of pH and the host-plant on the soil persistence of two alfalfa-nodulating rhizobia with different saprophytic and symbiotic characteristics

The acid tolerance of Sinorhizobium meliloti in culture media and in soils is considered a useful criteria to select for strains with improved survival in agricultural acidic soils. Using a glass tube system with gamma-irradiated soil at different pH values, we analysed the survival of two different alfalfa-nodulating rhizobia: S. meliloti (pH_limit for growth 5.6–6.0) and the acid-tolerant Rhizobium sp. LPU83, closely related to the strain Rhizobium sp. Or191 (pH_limit for growth below 5.0). Although the acid-tolerant rhizobia showed a slightly better survival during the first months in acid soil (pH=5.6), none of the strains could be detected 2 months after inoculation (bacterial counts were below 10³ colony-forming units (cfu)/30 g of soil). The inclusion of two alfalfa plants/glass tube with soil, however, supported the persistence of both types of rhizobia at pH 5.6 for over 2 months with counts higher than 9×10⁶ cfu/30 g of soil. Remarkably, in the presence of alfalfa the cell densities reached by S. meliloti were higher than those reached by strain LPU83, which started to decline 1 week after inoculation. Although more acid-sensitive in the culture medium than the Or191-like rhizobia, in the presence of the host plant the S. meliloti strains showed to be better adapted to the free-living condition, irrespective of the pH of the soil.     

Proton release by roots of Medicago murex and Medicago sativa growing in acidic conditions, and implications for rhizosphere pH changes and nodulation at low pH

Medicago murex nodulates faster and produces more nodules than Medicago sativa in acidic sandy soils. Experiments using a ‘root mat’ approach and videodensitometry examined pH changes in the rhizospheres of nitrate-fed plants of M. murex and M. sativa. Using the ‘root mat’ approach with soil disks of pH 4.49, M. sativa cv. Aquarius acidified its rhizosphere by approximately 0.2-0.4 pH-units within 4 d, while M. murex cv. Zodiac did not acidify its rhizosphere. Rates of H⁺ release were higher from M. sativa than from M. murex. Videodensitometry of roots embedded in agarose of pH 4.5 showed that the mature parts of the tap-root of both species exuded OH⁻ ions, but was approximately twofold more in M. murex than in M. sativa. Consequently, young parts of the M. sativa rhizosphere were less alkaline than that of M. murex. It is suggested that the difference in nodulation response between the two species at low pH may be related to the different patterns of rhizosphere acidification: the stronger rhizosphere acidification of M. sativa being less favourable for survival and growth of Sinorhizobium medicae. The higher rate of rhizosphere acidification by M. sativa roots may be related to its genetic characteristics including greater relative root growth rate and greater sensitivity to acidity in comparison to M. murex.     

Growth promotion of the seawater-irrigated oilseed halophyte Salicornia bigelovii inoculated with mangrove rhizosphere bacteria and halotolerant Azospirillum spp.

 Inoculation of the oilseed halophyte Salicornia bigelovii Torr. with eight species of halotolerant bacteria, grown in seawater-irrigated pots under environmental conditions native to the plant's habitat, resulted in significant plant growth promotion by the end of the growing season, 8–11 months later. Statistical analysis demonstrated that inoculation with Azospirillum halopraeferens, a mixture of two Azospirillum brasilense strains, a mixture of Vibrio aestuarianus and Vibrio proteolyticus, or a mixture of Bacillus licheniformis and Phyllobacterium sp. significantly increased plant height and dry weight at the end of the season. Some of the bacterial strains also increased the number of side branches and the size of the spikes. The bacteria did not affect the number of seeds or their weight. Inoculation with the mangrove cyanobacterium Microcoleus chthonoplastes had no effect on plant foliage variables. At the end of the growing season, the N and protein content of the plant foliage was significantly reduced by bacterial inoculation; however, the N and protein content of seeds significantly increased. The P content in foliage increased significantly in plants treated with all the bacteria except M. chthonoplastes, whereas the total lipid content of foliage increased significantly only when plants were inoculated with a mixture of A. brasilense strains or with M. chthonoplastes. In three inoculation treatments palmitic acid in seeds significantly increased and linoleic acid significantly decreased. This study demonstrates the feasibility of using bacteria to promote the growth of halotolerant plants cultivated for forage and seed production in proposed seawater-irrigated agriculture. Received: 3 January 2000     

The fate of markerAzospirillum lipoferum inoculated into rice and its effect on growth,yield and N2 fixation of plants studied by acetylene reduction,15N2 feeding and15N dilution techniques

Summary A spontaneous mutant ofAzospirillum lipoferum, resistant to streptomycin and rifampicin, was inoculated into the soil immediately before and 10 days after transplanting of rice (Oryza sativa L.). Two rice varieties with high and low nitrogen-fixing supporting traits, Hua-chou-chi-mo-mor (Hua) and OS4, were used for the plant bacterial interaction study. The effect of inoculation on growth and grain and dry matter yields was evaluated in relation to nitrogen fixation, by in situ acetylene reduction assay,¹⁵N₂ feeding and¹⁵N dilution techniques. A survey of the population of marker bacteria at maximum tillering, booting and heading revealed poor effectivety. The population of nativeAzospirillum followed no definite pattern. Acetylene-reducing activity (ARA) did not differ due to inoculation at two early stages but decreased in the inoculated plants at heading. In contrast, inoculation increased tiller number, plant height of Hua and early reproductive growth of both varieties. Grain yield of both varieties significantly increased along with the dry matter. Total N also increased in inoculated plants, which was less compared with dry matter increase.¹⁵N₂ feeding of OS4 at heading showed more¹⁵N₂ incorporation in the control than in the inoculated plants. The ARA,¹⁵N and N balance studies did not provide clear evidence that the promotion of growth and nitrogen uptake was due to higher N₂ fixation.     

Effects of antibiotic-producing Streptomyces on nodulation and leaf spot in alfalfa

The ability of antibiotic-producing streptomycetes to colonize alfalfa (Medicago sativa L.) plants and influence the activities of a fungal plant pathogen (Phoma medicaginis var. medicaginis) and a mutualistic symbiont (Sinorhizobium meliloti) was investigated. Streptomyces strains were introduced around seeds at the time of planting. Hyphal filaments and spore chains were observed by scanning electron microscopy on roots of alfalfa seedlings receiving the streptomycete amendment. Streptomyces strain densities on leaves decreased 10–100-fold over an 8-week period, while densities on roots remained constant over time. The Streptomyces strains also colonized alfalfa root nodules. We then tested the ability of 15 antibiotic-producing strains of Streptomyces to inhibit in vitro growth of Phoma medicaginis var. medicaginis Malbr. & Roum., the causal agent of spring blackstem and leaf spot of alfalfa. The majority of the Streptomyces strains inhibited growth of three diverse strains of P. medicaginis. In a detached leaf assay, one Streptomyces strain decreased leaf spot symptoms caused by P. medicaginis when inoculated onto leaves 24 h before the pathogen. Two Streptomyces strains decreased defoliation caused by P. medicaginis when the streptomycetes were introduced around seeds at the time of planting. We also examined inhibitory activity of Streptomyces strains against 11 strains of S. meliloti. Eight of the 15 Streptomyces strains inhibited in vitro growth of five or more of the S. meliloti strains, while four Streptomyces strains had no effect on growth of any test strains. In a growth chamber assay, two of six Streptomyces strains, when inoculated into the planting mix, significantly reduced plant dry weight compared to the treatment with S. meliloti alone, but did not significantly reduce the number of nodules. These results suggest that careful selection of Streptomyces isolates for use in biological control of plant diseases will limit the potential negative impacts on rhizobia.     

Interactions between mycorrhizal fungi and mycorrhizosphere bacteria during mineral weathering: Budget analysis and bacterial quantification

The impact of ectomycorrhizal fungi or rhizosphere bacteria on tree seedling growth and nutrient uptake is well known. However, few studies have combined those microorganisms in one experiment to clarify their relative contribution and interactions in nutrient acquisition. Here, we monitored the respective contributions of pine roots, two ubiquitous forest ectomycorrhizal fungi Scleroderma citrinum and Laccaria bicolor, and two S. citrinum-mycorrhizosphere bacterial strains of Burkholderia glathei and Collimonas sp., on mineral weathering, nutrient uptake, and plant growth. Pinus sylvestris plants were grown on quartz–biotite substrate and inoculated or not with combinations of mycorrhizal fungi and/or bacterial strains. Magnesium and potassium fluxes were measured and nutrient budgets were calculated. Both ectomycorrhizal fungi significantly increased Mg plant uptake. No significant effects of the two bacterial strains were detected on the K and Mg budgets, but co-inoculating the mycorrhizal fungus S. citrinum and the efficient mineral-weathering B. glathei bacterial strain significantly improved the Mg budget. Similarly, co-inoculating S. citrinum with the Collimonas sp. bacterial strain significantly improved the pine biomass compared to non-inoculated pine plants. In order to better understand this process, we monitored the survival of the inoculated bacterial strains in the quartz–biotite substrate, the pine rhizosphere, and the mycorrhizal niche. The results showed that the two bacterial strains harboured different colonization behaviours both of which depended on the presence of the ectomycorrhizal partner. The populations of the Burkholderia strain were maintained in all these environments with a significantly higher density in the mycorrhizal niche, especially of S. citrinum. In contrast the population of the Collimonas strain reached the detection level except in the treatment inoculated with S. citrinum. These results highlight the need for taking into account the ecology of the microorganisms, and more specifically the fungal–bacterial interactions, when studying mineral weathering and plant nutrition.     

Commercial Extract of Ascophyllum nodosum Improves Root Colonization of Alfalfa by Its Bacterial Symbiont Sinorhizobium meliloti

The soil bacterium Sinorhizobium meliloti forms a symbiotic relationship with alfalfa (Medicago sativa) roots, which results in the formation of intracellular root nodules. This symbiosis increases nitrogen (N) in the soil; however, to establish such a synergistic relationship, a complex communication system is required between the bacterium and its legume host. Rhizobacteria are known to respond to plant root exudates and produce signal molecules known as “Nod” factors. Research suggests that the brown seaweed (Ascophyllum nodosum) extract (ANE) stimulates both root nodulation and growth of alfalfa (Khan et al. 2011 Khan, W., Palanisamy, R., Critchley, A. T., Smith, D. L., Papadopoulos, Y. and Prithiviraj, B. 2011. Ascophyllum nodosum (brown seaweed) extract and its organic fractions stimulate root nodulation and growth of alfalfa (Medicago sativa). Communications in Soil Science and Plant Analysis (Accepted),  [Google Scholar]). To elucidate the mechanism of action, the effects of ANE on the early stages of root–rhizobia interactions were examined. A. nodosum extract (ANE) and its organic fractions were prepared and alfalfa roots were treated. After 2 days, the treated roots were inoculated with S. meliloti. The roots from treated plants were excised and observed for colony-forming units. To verify whether ANE elicited the synthesis and secretion of factors similar to those induced by luteolin, S. meliloti cultures were treated with ANE and the bacterial components were analyzed by high-pressure liquid chromatography (HPLC). To study Nod factor induction by S. meliloti due to ANE treatment, a root hair deformation assay was performed. A translational fusion of S. meliloti NodC:LacZ (strain JM57) was used to observe the effect of ANE on bacterial gene expression. When S. meliloti culture medium was supplemented with ANE, no effect on bacterial growth was observed. However, it was observed that the attachment of S. meliloti to the root hairs was improved. Similarly in vitro ANE root treatments, followed by S. meliloti inoculation, increased bacterial colonies. HPLC profiles and a root hair deformation assay suggested that ANE elicits production of compounds similar to the Nod factor, which are normally induced by the plant signaling molecule luteolin. The results suggest that ANE may contain compound(s) that promote the legume–rhizobia symbiotic relationship and plant signaling.     

Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp.

 Four experiments were performed under gnotobiotic conditions to select strains of the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. as inocula of rice plants. Eighty strains of H. seropedicae originally isolated from rice, sorghum and maize plants, were tested in test tube cultures with N-free agar as the substrate. Rice plants showed medium and high increases in their fresh weight in response to inoculation with nineteen strains. These strains were tested again, and six strains were then selected to evaluate their contribution to the N of the plant via biological N₂ fixation (BNF) using an agar growth medium containing 5 mg N l^–1of ¹⁵N-labelled (NH₄)₂SO₄. The contribution of the strains to plant N via BNF varied from 54% when rice plants were inoculated with strain ZAE94, to 31% when strain ZAE67 was used. These results were confirmed in the fourth gnotobiotic experiment, which also included strains of the new N-fixing bacteria belonging to the genus Burkholderia, isolated from rice, as well as a strain of Burkholderia vietnamiensis, isolated from rice rhizosphere. Burkholderia spp. strains showed similar effects to those observed for H. seropedicae strains, while B. vietnamiensis fixed only 19% of plant total N. The best four strains were tested in a pot experiment where pre-germinated, inoculated rice seedlings were grown in soil labelled with ¹⁵N. The results confirmed the gnotobiotic experiments, although the levels of N in the rice plants derived from BNF of the selected H. seropedicae and Burkholderia spp. strains were lower. Nevertheless, there was an increase in N content in grains of inoculated plants, and the results showed that the method used for strain selection is very useful and can be applied to other strains of N₂-fixing bacteria and plants. Received: 4 May 1999     

Inoculation responses of perennial forage legumes grown in fresh hill‐land ultisols as affected by soil acidity‐related factors

A growth chamber experiment was initiated with two field moist, marginal and acidic (pH 5.1–5.2) soils of the Lily series (Typic Hapludults) in order to determine the need for improved legume‐rhizobia symbioses for forage species of current, or potential, use in the renovation of Appalachian hill‐land pastures. One soil was from an abandoned pasture having broomsedge (Andropogon virginicus L.) as the predominant vegetation, whereas the other was from a minimally‐managed pasture dominated by orchardgrass (Dactylis glomerata L.). Treatments included inoculation (or no inoculation) and the addition of aluminum, nil, or lime to provide a range of soil acidities. Both soils contained effective populations of naturalized rhizobia for white clover (Trifolium repens L.) and red clover (Trifolium pratense L.), but low and/or ineffective naturalized populations of rhizobia for alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), bigflower vetch (Vicia grandiflora Scop.), and flatpea (Lathyrus sylvestris L.). Seed inoculation, by lime‐pelleting, was highly beneficial in establishing effective symbioses for all these latter species. The addition of low levels of aluminum or lime (1.5 and 2.0 cmol/kg soil, respectively) had little effect on any of the symbioses, with the exception of those for alfalfa. Thus, an improved legume rhizobia symbiosis would not seem to be a prerequisite for renovating pastures established on chemically similar ultisols with the forage legume species examined in this study, especially if the pasture has at least some history of management.