Response of introduced Bradyrhizobium strains infectinga promiscuous soybean cultivar

Two field experiments were established to assess the competitiveness of foreign bradyrhizobia in infecting the promiscuous soybean cultivar TGX 536-02D. Seeds were inoculated with antibiotic mutants of the bradyrhizobia strains before planting after land preparation. Soybean plants were harvested at pre-determined days after planting for estimating nodule number, nodule dry weight, nodule occupancy, shoot dry weight and seed yield. Results show that nodule number and dry weight significantly increased and showed great variability at 84 days after planting (DAP), probably due to differences in the ability of inoculant bradyrhizobia to form nodules with the soybean cultivar TGX 536-02D. Increased shoot dry weight, %N, total N and seed yield were a result of increased nodulation by the effective and competitive inoculant Bradyrhizobium strains. Strain USDA 110 occupied the highest percentage of nodule sites because it was more competitive than the other Bradyrhizobium strains. These results show that there was high potential for increasing growth and seed yield of the promiscuous soybean cultivar TGX 536-02D by inoculation with foreign Bradyrhizobium strains.     

<Emphasis Type="Italic">Mesorhizobium jarvisii</Emphasis> sv. astragali as predominant microsymbiont for <Emphasis Type="Italic">Astragalus sinicus</Emphasis> L. in acidic soils,Xinyang, China

Background and aims

Common bean (Phaseolus vulgaris L.) nodulates with a wide range of rhizobia. Amongst these is Bradyrhizobium, which is inefficient but able to induce profuse nodulation on this crop. Based on this observation, we tested whether co-inoculating bradyrhizobia with a more standard common bean symbiont, Rhizobium tropici, could stimulate growth and nodulation of common bean, thus contributing to a more effective symbiosis.

Methods

Rhizobium tropici was co-inoculated with two Bradyrhizobium strains applied at three different doses (10⁴, 10⁶, and 10⁸ CFU seed^?1) under sterile conditions, and at a single dose (10⁸ CFU seed^?1) in non-sterile soil. Plant biomass, nodulation, and N accumulation in plant tissues were evaluated.

Results

Co-inoculated plants produced more nodules, and accumulated more shoot dry biomass and nitrogen than plants inoculated with R. tropici alone under gnotobiotic conditions. Significant responses were observed at the highest inoculum dose and a significant correlation between dose and shoot dry weight was observed. Co-inoculation increased biomass and N accumulation in non-sterile soil, although with a smaller magnitude.

Conclusions

Altogether, our findings suggest that the co-inoculation with bradyrhizobia contributed to an improved symbiotic interaction between R. tropici and common beans.

     

Influence of 5-Methyltryptophan-Resistant Bradyrhizobium japonicum on Soybean Root Nodule Indole-3-Acetic Acid Content

Bradyrhizobium japonicum mutants resistant to 5-methyltryptophan were isolated. Some of these mutants were found to accumulate indole-3-acetic acid (IAA) and tryptophan in culture. In greenhouse studies, nodules from control plants inoculated with wild-type bradyrhizobia contained 0.04, 0.10, and 0.58 μg of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules⁻¹, respectively. Nodules from plants inoculated with 5-methyltryptophan-resistant bradyrhizobia contained 0.94, 1.30, and 10.6 μg of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules⁻¹, respectively. This manyfold increase in nodule IAA content indicates that the Bradyrhizobium inoculum can have a considerable influence on the endogenous IAA level of the nodule. Further, these data imply that much of the IAA that accumulated in the high-IAA-containing nodules was of bacterial rather than plant origin. These high-IAA-producing 5-methyltryptophan-resistant bacteria were poor symbiotic nitrogen fixers. Plants inoculated with these bacteria had a lower nodule mass and fixed less nitrogen per gram of nodule than did plants inoculated with wild-type bacteria.     

Hydrogen Recycling by Rhizobium leguminosarum Isolates and Growth and Nitrogen Contents of Pea Plants (Pisum sativum L.)

The ability to recycle H₂ evolved by nitrogenase is thought to be of importance in increasing the efficiency of N₂ fixation and to be a factor in increasing plant yield in symbiotic systems. To determine whether this ability is a significant factor in the Rhizobium leguminosarum-Pisum sativum L. system, plants were inoculated with R. leguminosarum isolates which differed in their ability to oxidize H₂ and in their relative efficiency of N₂ fixation. These plants were grown at three levels of irradiance and harvested after 3, 4, and 5 weeks of growth for determination of C₂H₂ reduction, H₂ evolution and uptake, plant dry weight, and N content. Plants inoculated with uptake hydrogenase-positive (Hup⁺) isolates did not exhibit higher dry weight or N content than those inoculated with Hup⁻ isolates under any of the growth conditions studied. The efficiency of the nitrogenase system of Hup⁻ isolates increased at a low irradiance, a factor which may allow them to compete successfully with Hup⁺ isolates. In some Hup⁺R. leguminosarum isolates, H₂ oxidation is coupled to ATP formation, whereas in others, it is not. There were no differences in plant dry weight and N content in plants inoculated with the two types and grown for 5 weeks at three irradiance levels. The addition of H₂ to Hup⁺ nodules whose supply of photosynthate had been removed by stem excision did not increase C₂H₂ reduction in either coupled or uncoupled types.     

Nitrogen-fixing potential of micropropagated clones of Acacia mangium inoculated with different Bradyrhizobium spp. strains

Five A. mangium seedlings of different shoot lengths were selected from a 600-seed screening experiment and micropropagated. Two-week-old rooted microcuttings of the 5 micropropagated clones were inoculated with 3 specific Bradyrhizobium spp. strains in 15 combinations. After 5 months of growth, nodule dry weight and shoot dry weight data showed significant effects of clone and Bradyrhizobium spp. strain. Clones RR-G₁ and IR-M₂ and Bradyrhizobium sp. Aust13c resulted in the highest dry-matter production and most efficient nodulation. No interaction was observed between clone and Bradyrhizobium spp. strain, which indicates that the Bradyrhizobium spp. strain and the host plant can be selected separately.     

Genetic Structure and Symbiotic Characteristics of a Bradyrhizobium Population Recovered from a Pasture Soil

We examined the genetic structure and symbiotic characteristics of Bradyrhizobium isolates recovered from four legume species (Lupinus albus [white lupine], Lupinus angustifolius [blue lupine], Ornithopus compressus [yellow serradella], and Macroptilium atropurpureum [sirato]) grown in an Oregon soil. We established that multilocus enzyme electrophoresis (MLEE) can provide insights into the genetic relatedness among Bradyrhizobium strains by showing a positive correlation (r² = ≥0.90) between the relatedness of Bradyrhizobium japonicum strains determined by MLEE at 13 enzyme loci and that determined by other workers using either DNA-DNA hybridization or DNA sequence divergence estimates. MLEE identified 17 electrophoretic types (ETs) among 95 Bradyrhizobium isolates recovered from the four hosts. Although the overall genetic diversity among the ETs (H = 0.69) is one of the largest measured to date in a local population of any soilborne bacterial species, there was no evidence of multilocus structure (linkage disequilibrium) within the population. The majority of the isolates (73%) were represented by two closely related ETs (2 and 3) which dominated the root nodules of white lupine, serradella, and siratro. In contrast, ET1 dominated nodules of blue lupine. Although representative isolates from all of the 17 ETs nodulated siratro, white lupine, blue lupine, and big trefoil (Lotus pedunculatus), they were either completely ineffective or poorly effective at fixing nitrogen on these hosts. Despite the widespread use of serradella as a surrogate host for lupine-nodulating bradyrhizobia, 7 of the 17 ETs did not nodulate this host, and the remaining 10 ETs were ineffective at fixing nitrogen.     

Isolation and Symbiotic Characteristics of Two Tn5-Derived Phage-Resistant <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> Strains that Nodulate Soybean

Using transponson Tn5 mutagenesis, two transconjugants of Bradyrhizobium japonicum with the properties of both phage resistance and ability to induce nodulation were isolated at the frequency of 0.02%. These transconjugants were tested for their symbiotic performance on soybean cv. JS335 under greenhouse and field conditions. Both phage-resistant mutants induced nodules (nod (+)), but the transconjugant B. japonicum E13 was ineffective in nitrogen fixation (fix (-)). Rhizobiophage presence in the inoculum of phage-resistant mutants did not influence the symbiotic effectiveness. The mixture of wild strain and phage in the inoculum caused reduced symbiotic performance under controlled conditions, while under a field environment phage (100 and 500 mul of approximately 10(8) particles ml(-1)) presence did not have any recognizable effect on increased nodule dry weight, nitrogenase activity, or foliar N(2) content. On the basis of restriction fragment length polymorphism analysis, phage-sensitive, less effective, homologous bradyrhizobia belonging to B. japonicum were detected in root nodules of both inoculated and uninoculated plants. Inoculation of a higher concentration of phage in the inoculum significantly reduced the symbiotic performance, while the lower concentration of phage did not show any effect on phage-susceptible, less effective, homologous bradyrhizobia or, thus, symbiotic efficiency under field conditions. The phage-resistant mutant B. japonicum A49 showed effective symbiosis as efficient as that of the wild strain. Inoculation of phage-resistant mutants with lytic phage may reduce the occupancy of phage-susceptible, ineffective/less effective/mediocre homologous bradyrhizobia strains under natural complex soil conditions.     

Inoculation Response of Legumes in Relation to the Number and Effectiveness of Indigenous Rhizobium Populations

The response of legumes to inoculation with rhizobia can be affected by many factors. Little work has been undertaken to examine how indigenous populations or rhizobia affect this response. We conducted a series of inoculation trials in four Hawaiian soils with six legume species (Glycine max, Vigna unguiculata, Phaseolus lunatus, Leucaena leucocephala, Arachis hypogaea, and Phaseolus vulgaris) and characterized the native rhizobial populations for each species in terms of the number and effectiveness of the population for a particular host. Inoculated plants had, on average, 76% of the nodules formed by the inoculum strain, which effectively eliminated competition from native strains as a variable between soils. Rhizobia populations ranged from less than 6 × 10⁰/g of soil to 1 × 10⁴/g of soil. The concentration of nitrogen in shoots of inoculated plants was not higher than that in uninoculated controls when the most probable number MPN counts of rhizobia were at or above 2 × 10¹/g of soil unless the native population was completely ineffective. Tests of random isolates from nodules of uninoculated plants revealed that within most soil populations there was a wide range of effectiveness for N₂ fixation. All populations had isolates that were ineffective in fixing N₂. The inoculum strains generally did not fix more N₂ than the average isolate from the soil population in single-isolate tests. Even when the inoculum strain proved to be a better symbiont than the soil rhizobia, there was no response to inoculation. Enhanced N₂ fixation after inoculation was related to increased nodule dry weights. Although inoculation generally increased nodule number when there were less than 1 × 10² rhizobia per g of soil, there was no corresponding increase in nodule dry weight when native populations were effective. Most species compensated for reduced nodulation in soils with few rhizobia by increasing the size of nodules and therefore maintaining a nodule dry weight similar to that of inoculated plants with more nodules. Even when competition by native soil strains was overcome with a selected inoculum strain, it was not always possible to enhance N₂ fixation when soil populations were above a threshold number and had some effective strains.     

Rhizobitoxine production and symbiotic compatibility of Bradyrhizobium from Asian and North American lineages of amphicarpaea.

Reciprocal inoculations with Bradyrhizobium sp. isolates from the North American legume Amphicarpaea bracteata (L.) Fern. (Phaseoleae-Glycininae) and from a Japanese population of its close relative Amphicarpaea edgeworthii (Benth.) var. japonica were performed to analyze relative symbiotic compatibility. Amphicarpaea edgeworthii plants formed few or no nodules with any North American bradyrhizobial strains isolated from A. bracteata, but all A. bracteata lineages formed effective nitrogen-fixing nodules with Japanese Bradyrhizobium isolates from A. edgeworthii. However, one group of A. bracteata plants (lineage Ia) when inoculated with Japanese bradyrhizobia developed a striking leaf chlorosis similar to that known to be caused by rhizobitoxine. The beta-cystathionase inhibition assay demonstrated that significant amounts of rhizobitoxine were present in nodules formed by these Japanese bradyrhizobia. No North American bradyrhizobial isolate from A. bracteata induced chlorosis on any plants, and the beta-cystathionase assay failed to detect rhizobitoxine in nodules formed by these isolates. The role of rhizobitoxine in A. edgeworthii nodulation development was tested by inoculating plants with a Bradyrhizobium elkanii rhizobitoxine-producing strain, USDA 61, and two mutant derivatives, RX17E and RX18E, which are unable to synthesize rhizobitoxine. Amphicarpaea edgeworthii inoculated with wild-type USDA 61 developed >150 nodules per plant, while plants inoculated with RX17E and RX18E developed fewer than 10 nodules per plant. Thus, efficient nodule development in A. edgeworthii appears to be highly dependent on rhizobitoxine production by Bradyrhizobium strains.     

Ectomycorrhizal symbiosis enhanced the efficiency of inoculation with two Bradyrhizobium strains and Acacia holosericea growth

Abstract Two strains of Bradyrhizobium sp., Aust 13C and Aust 11C, were dually or singly inoculated with an ectomycorrhizal fungus, Pisolithus albus to assess the interactions between ectomycorrhizal symbiosis and the nodulation process in glasshouse conditions. Sequencing of strains Aust 13C and Aust 11C confirmed their previous placement in the genus Bradyrhizobium. After 4 months culture, the ectomycorrhizal symbiosis promoted plant growth and the nodulation process of both Bradyrhizobium strains, singly or dually inoculated. PCR/RFLP analysis of the nodules randomly collected in each treatment with Aust 13C and/or Aust 11C: (1) showed that all the nodules exhibited the same patterns as those of the Bradyrhizobium strains, and (2) did not detect contaminant rhizobia. When both Bradyrhizobium isolates were inoculated together, but without P. albus IR100, Aust 11C was recorded in 13% of the treated nodules compared to 87% for Aust 13C, whereas Aust 11C and Aust 13C were represented in 20 and 80% of the treated nodules, respectively, in the ectomycorrhizal treatment. Therefore Aust 13C had a high competitive ability and a great persistence in soil. The presence of the fungus did not significantly influence the frequencies of each Bradyrhizobium sp. root nodules. Although the mechanisms remain unknown, these results showed that the ectomycorrhizal and biological nitrogen-fixing symbioses were very dependent on each other. From a practical point of view, the role of ectomycorrhizal symbiosis is of great importance to N₂ fixation and, consequently, these kinds of symbiosis must be associated in any controlled inoculation.     

A Host-specific, Nitrogen Fixation Mutant of Bradyrhizobium: Physiology on Three Host Plants

NC92 #284 is a transposon mutant of Bradyrhizobium sp. (Arachis)strain NC92 and has a host-specific fixation phenotype. It appearsto be ineffective on the host pigeonpea (90% reduction in shootN compared to that of the wild type), but partially effectiveon two other host plants, groundnut and siratro (50% and 20%reduction in shoot N compared to the wild type, respectively).To understand this phenomenon of host-specific fixation, thephysiological basis of the phenotypes was investigated. Host-dependentdifferences in symbiotic effectiveness were largely explainedby the degree to which nitrogenase activity was impaired inthe various #284 symbioses. Nodulation and the onset of nitrogenfixation were found to be delayed on all three hosts, but tothe greatest degree on pigeonpea. The specific activity of nitrogenaseper gram nodule was also reduced on all three hosts, again tothe greatest extent on pigeonpea. By contrast, the carbon costsand relative efficiencies of each symbiosis were similar tothe wild type. The results indicate that the host-specific fixationphenotype of #284 is reflected in a quantitative reduction inthe amount of N₂ fixed. Thus the phenotypes reflect the differentability of the three host plants to tolerate or support the#284 mutation, rather than a defect in a specific interactionbetween #284 and a particular host plant. Key words: Bradyrhizobium, nodules, nitrogenase activity, relative efficiency, Arachis hypogaea, Cajanus cajan, Macroptilium atropurpureum     

Nitrate Reductase in Wheat Plants Grown Under Water Stress and Inoculated with Azospirillum spp.

The present investigation has been performed to evaluate nitrate reductase (NR) and nitrogenase activities as well as growth and mineral nutrition of wheat plants grown under drought stress and inoculated with different Azospirillum strains (NR⁻ and NR⁺). Fresh, dry mass and water content decreased with decreasing soil moisture content, which was accompanied with low soluble sugars and soluble protein content and increase in the total amino acids content. Azospirillum inoculation with either bacterial strain (NR⁻ and NR⁺) significantly increased the above characteristics even at 40 % moisture content. NR activity decreased in both the shoots and roots by decreasing soil moisture content. NR⁺ strain exhibited increased root NR activity compared with uninoculated plants or inoculated with NR⁻ strain. However, plants inoculated with NR⁻strain increased NR activity in the shoot more than in the root of the same plant and in the shoot of control plants. Inoculation with either NR⁻ and NR⁺ Azospirillum strains gave higher nitrogenase activity than uninoculated control plants. The low N supply (0.5 mM) did not affect nitrogenase activity. NR⁻strain was less effective than NR⁺strain in promoting total N-yield, spike numbers and their mass per pot. Azospirillum inoculation exhibited no significant changes in wheat Mg²⁺ content. However, K⁺ and Ca²⁺ have shown significantly increased values. Azospirillum beneficial effect on plant N balance and growth are most probably composed of multiple mechanisms and beneficial NR is one of them. The importance of Azospirillum NR⁺strains for increasing wheat resistance to water stress is also supported by the obtained data. This revised version was published online in July 2006 with corrections to the Cover Date.     

Relative Efficacy of Different Alfalfa Cultivar-Rhizobium meliloti Strain Combinations for Symbiotic Nitrogen Fixation

Five Rhizobium meliloti isolates known to have different capabilities for expression of nitrogenase activity under symbiotic conditions were used to inoculate four representative Medicago sativa cultivars under aseptic conditions. Nitrogenase activities and respiratory activity were measured for whole plants and excised nodules. Dry weights and nodule numbers were also recorded after 4 weeks of growth in plastic pouches on a nitrogen-free nutrient medium. Hydrogen evolution and acetylene reduction rates were used to calculate the fraction of reducing power allocated to dinitrogen reduction. Although the efficiency of the system defined in this way was poorly correlated with plant yield, a very high linear correlation was obtained between yield and the algebraic product of nitrogenase activity and efficiency. High correlation (r > 0.78) was obtained between respiration and nitrogenase activity for whole plants as well as for excised nodules. Nodular respiration accounted for between 10 and 20% of the total plant dark respiration. The four test cultivars exhibited significantly different symbiotic responses to the inocula, although trends in potential for expression of the nitrogenase system by the five R. meliloti strains were evident. There was significant interaction between the host plant and symbiont in determining nitrogenase activity and yield. This screening method allows quantitative discrimination between effective and ineffective host-inoculum combinations.     

Does oxygen limit nitrogenase activity in soybean exposed to elevated CO2?

Soybean (Glycine max L. Merr) plants grown under control (360 µmol mol^?1) or elevated CO₂ concentration (800 µmol mol^?1) from 33 to 42 d after sowing were assayed for various components of in vivo nitrogenase activity to test the hypothesis that increasing carbohydrate supply to nodules would increase the potential (i.e. O₂ saturated) nitrogenase activity and impose a more severe O₂ limitation on both nodule metabolism and total nitrogenase activity. Within 51 h of elevated CO₂ treatment, significant increases relative to control plants were seen in total nitrogenase activity expressed per plant. After 6 d of elevated CO₂, the total nitrogenase activity per plant was 18% higher than that in control. This was attributed to an initial increase in nodule size, and a subsequent increase in nodule number following plant exposure to elevated CO₂. However, after 9 d of elevated CO₂, the potential and total nitrogenase activities per gram nodule dry weight were lower, not higher than corresponding values in plants in the control treatment. These results did not support the hypothesis. It was concluded that the metabolic capacity of the control nodules were not limited by carbohydrate supply, at least at the assay temperatures employed here.     

Enhancement of Green Gram Nodulation and Growth by Bacillus Species

Rhizobacteria belonging to Bacillus sp. were isolated from the rhizosphere of green gram (Vigna radiata). Seed inoculation with the rhizobacteria showed stunting effect on root growth whereas four Bacillus strains caused stimulation of shoot growth at both 4 and 7 d of observations. Coinoculation of some Bacillus strains with effective Bradyrhizobium strain S24 resulted in enhanced nodulation and plant growth of green gram. The shoot dry mass (ratio to uninoculated control) varied from 1.32 to 6.33 at day 30 and from 1.28 to 3.55 at day 40 of plant growth. Nodule promoting effect after 40 d of plant growth was observed with majority of Bacillus strains except for MRS9 and MRS26. Maximum gains in nodulation, nitrogenase activity and plant growth were observed with Bacillus strains MRS12, MRS18, MRS22 and MRS27 after 40 d of plant growth, suggesting the usefulness of introduced rhizobacteria in improving crop productivity.     

Rhizobial hitchhikers from Down Under: invasional meltdown in a plant–bacteria mutualism?

Aim This study analysed the diversity and identity of the rhizobial symbionts of co‐existing exotic and native legumes in a coastal dune ecosystem invaded by Acacia longifolia. Location An invaded coastal dune ecosystem in Portugal and reference bradyrhizobial strains from the Iberian Peninsula and other locations. Methods Symbiotic nitrogen‐fixing bacteria were isolated from root nodules of plants of the Australian invasive Acacia longifolia and the European natives Cytisus grandiflorus, Cytisus scoparius and Ulex europaeus. Total DNA of each isolate was amplified by polymerase chain reaction (PCR) with the primer BOX A1R. Subsequent PCR‐sequencing and phylogenetic analyses of the internal transcribed spacer region and the nifD and nodA genes were performed for all different strains. Results The four plant species analysed were nodulated by bacteria from three different Bradyrhizobium lineages, although most of the isolates belonged to the Bradyrhizobium japonicum lineage sensu lato. Ninety‐five per cent of the bradyrhizobia isolated from A. longifolia, C. grandiflorus and U. europaeus in the invaded ecosystem had nifD and nodA genes of Australian origin. Seven isolates obtained in this study define a new distinctive nifD group of Bradyrhizobium from western and Mediterranean Europe. Main conclusions These results reveal the introduction of exotic bacteria with the invasive plant species, their persistence in the new geographical area and the nodulation of native legumes by rhizobia containing exotic symbiotic genes. The disruption of native mutualisms and the mutual facilitation of the invasive spread of the introduced plant and bradyrhizobia could constitute the first report of an invasional meltdown documented for a plant–bacteria mutualism.     

Effects of nickel on Frankia and its symbiosis with Alnus glutinosa (L.) Gaertn

The tolerance of nickel by Frankia in culture and in symbiosis with Alnus was determined. Yield of three Frankia strains was not affected significantly by 2.25 mM nickel when cultured in propionate medium containing hydolysed casein as nitrogen source. Yield of two strains in medium without combined nitrogen, and thus reliant on fixed nitrogen, was stimulated markedly by the same nickel concentration. Utilisation of nickel for synthesis of uptake hydrogenases is presumed to be the cause of enhanced nitrogenase activity.Although growth was reduced, treatment of 2-month-old seedlings with 0.025 mM nickel for 4 weeks did not affect nodulation significantly while nitrogenase activity was doubled. Nodulation and nitrogenase activity of seedlings receiving 0.075 mM nickel were inhibited markedly, while 0.5 mM nickel was lethal to all seedlings after 4 weeks of treatment. A few small, ineffective nodules were initiated early on some of the latter seedlings, suggesting that effects of nickel on host plant processes rather than Frankia are the primary cause of inhibition of nodulation. This interpretation is supported by the retention of substantial nitrogenase activity in 10-month-old plants 1 day after the treatment with 0.59 mM nickel, when the nickel content of roots and nodules was already maximal. No nitrogenase activity was detected after 3 days, by which time the leaves were almost completely necrotic. Over a 4 day period, most nickel was retained in the roots and nodules. Supplying histidine simultaneously at concentrations equal to, or in excess of, nickel prevented wilting and leaf necrosis, but did not increase translocation of nickel to the shoot.     

Colonization of root tissues and protection against Fusarium wilt of rye (Secale cereale) by nonpathogenic rhizosphere strains of Fusarium culmorum

Colonization of rye (Secale cereale) tissues by nonpathogenic rhizosphere Fusarium culmorum isolates DEMFc2 and DEMFc5 and a pathogenic strain DEMFc37, and their effect on plant fresh weight were studied in pot experiments. Both rhizosphere isolates colonized the epidermis and the cortex but were not found in vessels, while the pathogen colonized all three layers of root cells. The numbers of pathogen CFU isolated from plant tissues were much higher than those of the rhizosphere isolates in spite of the same number of macroconidia used as inoculum (1 × 10⁵ g⁻¹ of soil). Inoculation of seedlings with DEMFc2 resulted in a 20% increase, with DEMFc5 in more than a 20% reduction, and with DEMFc37 in a 38% reduction of shoot fresh weight of 14-day-old plants. Pre-colonization of plants with (either of) the rhizosphere isolates and subsequent inoculation with the pathogen resulted in plant weights the same as those observed in plants inoculated with the rhizosphere strain alone. The disease severity index for shoots of plants pre-colonized with DEMFc2 was reduced from class 4 (86% diseased plants) observed for plants inoculated with the pathogen alone to class 2 (average of 8% diseased plants) when pre-treated with the rhizosphere strain. The CFU number of the pathogen isolated from the interior of roots of plants pre-colonized with the rhizosphere isolates was as low as 10% of the number isolated from plants inoculated with the pathogen alone. A study of in vitro interactions between the rhizosphere isolates and the pathogen suggests that changes in plant colonization by the pathogen and its effect on fresh weight of plants pre-colonized with the rhizosphere isolates were not connected with inhibition of its growth by a direct action of the rhizosphere isolates. The results suggest that strain DEMFc2 can be considered as a potential biocontrol agent.     

Nitrogenase activity and N2 fixation are stimulated by elevated CO2 in a tropical N2-fixing tree

 Seeds of Gliricidia sepium, a fast-growing woody legume native to seasonal tropical forests of Central America, were inoculated with N₂-fixing Rhizobium bacteria and grown in environmentally controlled glasshouses for 67–71 days under ambient CO₂ (35 Pa) and elevated CO₂ (70 Pa) conditions. Seedlings were watered with an N-free, but otherwise complete, nutrient solution such that bacterial N₂ fixation was the only source of N available to the plant. The primary objective of our study was to quantify the effect of CO₂ enrichment on the kinetics of photosynthate transport to nodules and determine its subsequent effect on N₂ fixation. Photosynthetic rates and carbon storage in leaves were higher in elevated CO₂ plants indicating that more carbon was available for transport to nodules. A ¹⁴CO₂ pulse-chase experiment demonstrated that photosynthetically fixed carbon was supplied by leaves to nodules at a faster rate when plants were grown in elevated CO₂. Greater rates of carbon supply to nodules did not affect nodule mass per plant, but did increase specific nitrogenase activity (SNA) and total nitrogenase activity (TNA) resulting in greater N₂ fixation. In fact, a 23% increase in the rate of carbon supplied to nodules coincided with a 23% increase in SNA for plants grown in elevated CO₂, suggesting a direct correlation between carbon supply and nitrogenase activity. The improvement in plant N status produced much larger plants when grown in elevated CO₂. These results suggest that Gliricidia, and possibly other N₂-fixing trees, may show an early and positive growth response to elevated CO₂, even in severely N-deficient soils, due to increased nitrogenase activity. Received: 27 February 1996 / Accepted: 19 June 1996     

Inoculation and production of container-grown red alder seedlings

Summary The inoculation ofAlnus rubra (red alder) withFrankia sp. can lead to a highly efficient symbiosis. Several factors contribute to the successful establishment of nitrogenfixing nodules: (1) quantity and quality ofFrankia inoculant; (2) time and method of inoculation; (3) nutritional status of the host plant.Frankia isolates were screened for their ability to nodulate and promote plant growth of container-grown red alder. Inoculations were performed on seedlings and seeds. Apparent differences in symbiotic performance could be seen when seeds or seedlings were inoculated. Plants inoculated at planting performed significantly better than those inoculated four weeks later in terms of shoot height, nodule number and shoot dry weight. If inoculation was delayed further, reduction in shoot height, nodule number and shoot dry weight resulted. The effect of fertilizer was also investigated with regard to providing optimal plant growth after inoculation. Plants receiving 1/5 Hoagland's solution minus nitrogen showed maximal plant growth with abundant nodulation. Plants receiving 1/5 Hoagland's solution with nitrogen showed excellent plant growth with significantly reduced nodulation.