Inadequate iron supply and high bicarbonate impair the symbiosis of peanuts (Arachis hypogaea L.) with different Bradyrhizobium strains

In the present study, we examined the effects of iron deficiency in an acid solution and in an alkaline solution containing bicarbonate on the growth and nodulation of peanuts inoculated with different bradyrhizobial strains or supplied with fertilizer nitrogen.Inadequate iron supply in acid solution decreased the number of nodule initials, nodule number and nodule mass. Alleviating the iron deficiency increased acetylene reduction but not bacteroid numbers in nodules. Nitrogen concentrations in shoots of inoculated plants increased as iron concentrations in solution increased when determined at day 30 but not at day 50. Higher iron concentrations in solution were required for maximum growth of plants reliant on symbiotic nitrogen fixation than for those receiving fertilizer nitrogen.Adding bicarbonate to the solution with 7.5 M Fe markedly depressed nodule formation. This effect was much more severe than that of inadequate iron supply alone. Bicarbonate also decreased nitrogenase activity but did not decrease bacteroid concentrations in nodules.Both NC92 and TAL1000 nodulated peanuts poorly when bicarbonate was present. However, an interaction between iron concentrations in acid solutions and Bradyrhizobium strains on nodulation of peanuts was observed. Alleviating iron deficiency increased the number of nodule initials and nodules to a much greater extent for plants inoculated with TAL1000 than for plants inoculated with NC92.     

Nodulation ofMedicago sativa in solution culture

Summary When nitrate was maintained in continuous supply in solution cultures at concentration 0.02 mM to 2 mM it reduced the numbers of curled root hairs and of nodules. But the addition of indole-3-acetate with the inoculum overcame the nitrate-inhibition of curling without affecting nodulation; and there was no quantitative connection between numbers of curled hairs and of nodules when nitrate treatments of limited duration were applied at different times in relation to inoculationNitrate reduced nodule number to some extent even if present only before inoculation, or only on the first, second or third day after inoculation, so that reduction in nodule number was difficult to attribute solely or chiefly to interference with any one phase of the nodulation process. However, nitrate inhibited formation of infection threads and augmented the proportion of arrested infection threads, to such an extent that this could be important in limiting nodule number.Nitrate also delayed nodulation. The delay occurred early after inoculation and corresponded with delay in the appearance of infection threads.     

Effects of continuous nitrogen application and nitrogen preconditioning on nodulation and growth ofCeanothus griseus varhorizontalis

Rooted cuttings ofCeanothus griseus varhorizontalis were irrigated with 0, 10, 20, 50, 75 or 100ppm nitrogen as NH₄NO₃ for eight weeks prior to inoculation with infectiveFrankia. After inoculation, half of the plants for each treatment nitrogen level continued to be irrigated with the preconditioning nitrogen level and half were given no more supplemental nitrogen. For plants continuously receiving nitrogen, nodule initiation (nodule number) was inversely correlated with increasing supplemental nitrogen levels, and suppressed above 50 ppm N. Leaf nitrogen above 2% in continuous-N plants correlated with greatly reduced or suppressed nodulation. Plants maintained after inoculation without supplemental nitrogen showed influence of the prior nitrogen treatment on nodulation. Preconditioning at 50 ppm and above greatly reduced the number of nodules formed. The evidence suggests that stored internal nitrogen can regulate nodulation.Plant biomass accumulated maximally when nodulation was suppressed, at 75 and 100 ppm supplemental N applied continuously. Internode elongation during the nodulation period occurred only on nodulated plants, or in the presence of supplemental N (10 ppm and above).     

Nodulation and growth ofPhaseolus vulgaris in solution culture

Summary Conditions and techniques for achieving good nodulation ofPhaseolus vulgaris L. in continuously aerated solution were developed from greenhouse experiments.If nodules had been established, their growth and activity and the growth of the plant were at least as good in solution culture as in gravel culture. Nodule formation was observed within 10 days of inoculation in small volumes of solution culture (1 liter). In large volumes (19 liters), similarly prompt nodulation occurred only if the plants were inoculated before or immediately after the seedlings were transferred to the solution from gravel or vermiculite; and the nodules were restricted to the roots that had been present at the time of transfer. Delayed inoculation, 2 days after transfer to large volume solutions, led to sparse nodulation observed only after 3 weeks. Delay or inhibition of nodulation in large volumes of solution could not be explained by failute of bacteria to colonize roots or by sparsity of root hairs.Nodule initiation in solution culture was severely inhibited at pH below 5.4. An additional problem in growing N₂-dependent bean in solution culture was the buildup of Cl^– to toxic levels in the plant in nitrate-free media, even at solution concentrations as low as 0.4 mM Cl^–. Daily addition of 0.5 to 1.0 mg N per plant delayed nodule growth and activity slightly, but increased plant growth and alleviated the severe N-deficiency that otherwise developed before the onset of N₂-fixation.     

Response of Leucaena/Rhizobium symbiosis to mineral nutrients in Southwestern Nigeria

Pot and field experiments carried out at the International Institute of Tropical Agriculture (IITA) and at Fashola, Southwestern Nigeria, examined the effect of inoculation and N, P and micronutrients on nodulation and growth ofLeucaena leucocephala (Lam.) de Wit. In pot studies all parameters measured, except the percentage of nitrogen in shoots, were improved by inoculation, nitrogen and phosphorus. Micronutrients increased only nitrogen and allantoin contents. Interactions between inoculation and P, N and micronutrients on nodulation and growth of leucaena were observed. The effect of inoculation and fertilization with phosphorus or micronutrients was further investigated in field experiments. Establishment of uninoculated and unfertilized leucaena was poor at both locations due to low soil fertility and the presence of only a few native leucaena rhizobia. At one site, only inoculated plants were nodulated, while at the other, all plants produced nodules. Shoot dry weight, total nitrogen and phosphorus of inoculated plants were statistically equal to nitrogen-fertilized plants. Uninoculated plants were stunted. Generally, micronutrients did not influence nodulation, total nitrogen or growth of leucaena. They had only a positive effect on nitrogenase activity. Phosphorus increased total nitrogen and phosphorus uptake and plant growth. A 75% increase in shoot dry weight was obtained when 80 kg P ha^–1 was applied to inoculated leucaena with Rhizobium strain IRc 1045. Inoculated plants contained more allantoins than uninoculated ones but no significant correlation was found between these compounds and other parameters of N fixation.     

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.     

A comparison of the effect of combined nitrogen on nodulation in non-legumes and legumes

Summary 1. The effect of the presence of combined (ammonium) nitrogen in the rooting medium on nodule development has been investigated inAlnus glutinosa andMyrica gale, the plants being grown by a water culture technique with provision for the control of the level of combined nitrogen and of pH. For purposes of comparison a similar experiment with a legume (Ulex europaeus) has been included.2. In some instances the differential treatment with combined nitrogen was commenced and inoculation effected at an early stage in plant development, while in others the plants were grown on to a larger size before these treatments were applied.3. In the two non-legumes the presence of combined nitrogen led to an increase in the weight of nodulus formed per plant, at least at the lower levels of nitrogen. Relative to the enhanced growth of the plant as a whole, nodule development was continuously depressed as the level of combined nitrogen was increased.4. InUlex when the nitrogen status of the plants at the time of inoculation was similar to that in the corresponding non-legume experiment, the above responses were also shown by the legume. It is concluded thatAlnus andMyrica resemble legumes closely in their responses to combined nitrogen.5. The data show that inAlnus the effect of a given level of combined nitrogen on nodulation depends on the nitrogen status of the plant at the time of inoculation, the adverse effect tending to be stronger if the plant is initially relatively high in nitrogen. This suggests that as in legumes the effect of the combined nitrogen is exerted internally in the plant tissues.     

Production and function of jasmonates in nodulated roots of soybean plants inoculated with Bradyrhizobium japonicum

Little is known regarding production and function of endogenous jasmonates (JAs) in root nodules of soybean plants inoculated with Bradyrhizobium japonicum. We investigated (1) production of jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) in roots of control and inoculated plants and in isolated nodules; (2) correlations between JAs levels, nodule number, and plant growth during the symbiotic process; and (3) effects of exogenous JA and OPDA on nodule cell number and size. In roots of control plants, JA and OPDA levels reached a maximum at day 18 after inoculation; OPDA level was 1.24 times that of JA. In roots of inoculated plants, OPDA peaked at day 15, whereas JA level did not change appreciably. Shoot dry matter of inoculated plants was higher than that of control at day 21. Chlorophyll a decreased more abruptly in control plants than in inoculated plants, whereas b decreased gradually in both cases. Exogenous JA or OPDA changed number and size of nodule central cells and peripheral cells. Findings from this and previous studies suggest that increased levels of JA and OPDA in control plants are related to senescence induced by nutritional stress. OPDA accumulation in nodulated roots suggests its involvement in "autoregulation of nodulation."     

The effect of cobalt on the growth of young lucerne on a siliceous sand

Summary The nodulation and growth of young lucerne plants on a moderately acid siliceous sand were greatly increased by inoculation and the application of lime. In addition cobalt treatment significantly increased the yield of nitrogen per nodulated plant and the amount of nitrogen fixed per nodulated plant but had no effect on non-nodulated plants. Cobalt significantly increased the fresh weight of nodules per plant, due to greater nodule size. Furthermore the amount of nitrogen fixed per unit fresh weight of nodular tissue was substantially increased. The effects of cobalt on symbiotic nitrogen fixation led to significant increases (29 to 77 per cent) in the dry-matter yield of lucerne tops.     

Nodulation and nitrogen fixation in Purshia: inoculation responses and species comparisons

Summary Nitrogen fixing trees and shrubs may be useful in revegetation efforts. Speculation that insufficient endophyte populations in surface soils may limit non-leguminous symbiotic nitrogen fixation in marginal land was explored.Purshia tridentata andP. glandulosa seedlings were grown in greenhouse trials using ten soils from nativePurshia sites. Treatments include a control, an inoculated treatment, and six mmole nitrogen amendment. When inoculated with aP.tridentata crushed nodule inoculum, two of five non-nodulating soils and three sparsely nodulating soils produced well nodulated plants. Inoculation also increased nodule mass, total nitrogen, nitrogen content and shoot dry mass in plants from some of the soils. Of the three soils failing to produce nodulated plants when inoculated, one produced plants that responded well to nitrogen additions but failed to nodulate under low nitrogen conditions; another produced severely stunted plants indicating nutritional limitations on the host; and the third produced plants that were not nitrogen deficient. An application of nitrogen completely suppressed nodulation in all but one soil. The twoPurshia species were similar in nodulation, nitrogen fixation and growth, although important exceptions exist that indicate species may differ in adaptability to certain soil conditions.     

Nodulation of Trifolium repens at low pH by single and paired strains of Rhizobium leguminosarum biovar trifolii

Detailed individual nodulation profiles were obtained for five strains of Rhizobium leguminosarum biovar trifolii inoculated onto roots of Trifolium repens seedlings growing on an agar medium of pH 4.5. The time of appearance and the location of every nodule were noted for a period of 10 days after inoculation. Using these nodulation frequency profiles, pairings of strains were identified and six mixed-strain inoculation (1:1 ratio) experiments were subsequently performed at pH 4.5. Results from the mixed-inoculum experiments showed that the performance of a Rhizobium strain in single culture could not be reliably used to predict the outcome of a paired-inoculation study and that some seedlings were exclusively nodulated by rhizobia that performed poorly at low pH in single-culture inoculations. Received: 26 November 1996 / Accepted: 18 April 1997     

Regulation of nodulation in Alnus incana-Frankia symbiosis

We have studied regulation of nodulation in Alnus incana (L.) Moench using double inoculations in plastic pouches and a slide technique to observe root hair deformation. Initially, the distribution of nodules between main and lateral roots appeared quite constant, independent of the concentration of inoculum (1 to 250 μg of crushed nodules plant⁻¹). Susceptibility to infection after the second inoculation was restricted to lateral roots after the initial infections developed. When pre-existing nodules were excised before the second inoculation, subsequent nodules appeared to arise where infections had arrested at stages earlier than actual nodule emergence. We observed that root hairs formed postinoculation were very crowded and short with a pronounced deformation. No nodules were found later on this region of the root, suggesting a loss of susceptibility in this region. Split-root experiments with delays between inoculation of the first and second side of the root system showed irreversible, systemic inhibition of nodulation on the second side starting between 3 and 6 days after the inoculation of the first side. Only when compatible, infective strains were used in the first inoculation, was nodule formation inhibited after the second inoculation. We conclude that autoregulation of nodulation operates in Alnus incana and on a time scale similar to what is found in some legumes.     

Rhizobium infection and nodule development in soybean are affected by exposure of the cotyledons to light

The initiation of Rhizobium infections and the development of nodules on the primary root of soybean Glycine max L. Merr cv Williams seedlings are strongly affected by exposure of the cotyledons/hypocotyls to light. Seedlings in plastic growth pouches were inoculated with R. japonicum in dim light and the position of the root tip of each seedling was marked on the face of the pouch. The pouches were covered and kept in the dark for various times before exposing the upper portions of the plants (cotyledons and hypocotyls) to light. Maximum nodulation occurred if the plants were kept in the dark until 1 day after inoculation. The exposure of plants to light 2 days before inoculation reduced the number of nodules by 50% while the number of nodules was reduced by 70% if the plants were kept in the dark until 7 days after inoculation. Anatomical studies revealed that exposure to light prior to inoculation reduced both the number of infection centers with visible infection threads and the number of infections which developed nodule meristems. Plants kept in the dark for 7 days after inoculation formed a normal number of infection threads above the root tip mark, but very few of these infections developed a nodule meristem. It appears that light stimulates soybean to produce substances which can both inhibit the formation of infection threads and enhance the development of nodules from established infection threads. The effects of light on nodulation appear to be expressed independently of the Rhizobium-induced suppression of nodule formation in younger regions of the root.     

The use of MES buffer in early nodulation studies with annual Medicago species

The usefulness of 2-(N-morpholino)ethanesulfonic acid (MES) to stabilize the pH of solutions in the range pH 5.5 to 7.0 during early growth and nodulation of annual Medicago (medic) species was investigated.In the first experiment, effects of MES concentrations (0, 1, 2, 5, 10 mM) on growth and nodulation of Medicago polymorpha L. and the stability of solution pH were investigated. In the second experiment we assessed the effect of MES on the growth and nodulation of three medic species (M. truncatula Gaernt., M. polymorpha and M. murex Willd.) at a range of starting pH levels (5.5, 6.0, 6.5 and 7.0) with different concentrations of MES buffer (0, 0.5, 10 mM). In a third experiment, the effects of the concentration of extra cations and the species (K-ion or Na-ion), used as hydroxide to bring solutions containing different MES concentrations to target pH were investigated.MES had no effect on growth at MES concentrations as high as 10 mM but nodule numbers were increased by concentrations of MES of 2 mM or greater. The presence of extra K-ion or Na-ion in solutions had no effect on plant growth or nodulation. With low MES concentrations (0.5 mM), solution pH stability was improved by daily titration and the use of a high nitrate to ammonium ratio (9 : 1) in solution. This combination of strategies maintained pH within a narrow range without effects on annual medic growth or nodulation.     

The Development and Significance of the Root Nodules of Casaurina

Young plants of Casuarina cunninghamiana and of C. equisetifoliagrowing in water culture developed macroscopic nodules in 26to 35 days from inoculation, the nodulation being most successfulat pH near to neutrality and falling off much more rapidly atlower pH than in other non-legume nodule-forming genera, confirmingthe distinctiveness of the Casuarina organism. The roots springingfrom the nodule lobes are shown to be characterized by upwardgrowth and in this to resemble remarkably closely the correspondingroots of Myrica. Nodulated plants of Casuarina are able to growvigorously in culture solution free of combined nitrogen, showingthat fixation of atmospheric nitrogen occurs, amounting in C.cunninghamiana to 50 mg. per plant during 6 months of activegrowth. The evidence indicates that the fixation occurs in thenodules, and that these have exactly the same functional significanceas those of legumes.     

Nodulation and growth ofLeucaena leucocephala (Lam.) de Wit as affected by inoculation and N fertilizer

Leonard jar, pot and field experiments examined the effects of inoculation and the influence of nitrogen fertilizer on nodulation, nitrogen fixation and growth ofLeucaena leucocephala (Lam.) de Wit at IITA, Ibadan, Nigeria. Leucaena responded to both inoculation and/or nitrogen application. Shoot growth and total N and P of inoculated plants were comparable to those of the highest N treatment, and the values were about 55% greater than those of uninoculated ones. Field data indicated that toal N yields of inoculated leucaena were increased by 50% with 40 or 80 kg ha^–1 of N fertilizer. However, N fertilizer depressed N fixation by 56% as was expected from nodule mass data. N-fixation was delayed for about 8 weeks in the plots without N. Application of small amounts of N starter (20 ppm) proved to be beneficial to satisfy the plant need during the early stage of leucaena growth. The rhizobial strains IRc 1045 and IRc 1050 were effective, competitive and survived well in the field one year after their establishment.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

The effect of level of CaCO3, inoculation and lime pelleting on the nodulation and growth of beans in five acid soils

Summary The effect of level of CaCO₃, inoculation and lime pelleting on the nodulation, dry matter yield and % N content of common bean plants (Phaseolus vulgaris) grown in five acid soils was investigated in a greenhouse study. The soils represented a range in pH from 3.9 to 5.1, in exchangeable Al from 0.0 to 4 meq/100 gm, in exchangeable Mn from 0.35 to 2.32 me/100 gm, and in %C from 0.69 to 5.60.Nodule weight decreased with increasing %C and for the soil with highest %C (5.60) no nodules were observed. In soils with low organic matter and low exchangeable Al and Mn, inoculation increased nodule weight, dry matter yield and %N especially at the lowest pH level. Where the seeds were not inoculated, nodule weight and dry matter yield increased with soil pH. No such increases were observed where the seeds were inoculated. There was no apparent advantage in lime pelleting in such soils.In soils with low organic matter content and with substantial amounts of Al and/or Mn, liming increased nodule weight and dry matter yield, and decreased exchangeable Al and/or Mn. Lime pelleting was superior to mere inoculation in increasing nodule weight particularly at low lime rates.In soils with relatively high organic matter content, nodulation was very low or none at all. Low lime rates had little effect on exchangeable Al and Ca and dry matter yield. Higher lime rates, however, decreased exchangeable Al and dry matter yield but increased exchangeable Ca.     

Autoregulation of soybean nodulation: Delayed inoculation increases nodule number

The influence of seedling age at the time of inoculation on the regulation of nodule number in soybean (Glycine max [L.] Merr.) was examined in cv. Williams 82 and its hypernodulating mutant NOD1-3. Nodulation was evaluated on plants grown in plastic growth pouches or in vermiculite in 50- or 500-ml glass containers in growth chamber studies. Seeds or seedlings were inoculated once with Bradyrhizobium japonicum strain USDA 110 (10^k cells seedling^?1) between 0 and 15 days after sowing at 3- or 5-day intervals and were grown for 21 days after inoculation. Nodule number plant^?1 was similar across inoculation times in plants grown in growth pouches, but was significantly greater when inoculation was delayed and plants were grown in vermiculite in 500-ml containers. Plant culture in vermiculite in 50- or 500-ml containers confirmed the suppressive effect of restricted space for root growth on nodulation. Inoculation with 10⁵ or 10⁹ USDA 110 cells revealed that nodulation was inhibited by a high inoculum dose. There was a large increase in nodule number plant^?1 when plants were transferred from a restricted rooting environment (growth pouch culture) to a nonrestricted rooting environment (2-1 hydroponic pots). Autoregulation was also examined in split-root assemblies of plants in 500-ml containers of vermiculite. Controls involved concurrent inoculation of both root halves at 0. 4 or 8 days after transplant. Treatments involved time-separated inoculations of root halves with the primary and secondary inoculations being separated by 4 days. Plants were harvested at 21 days after inoculation. Williams 82 exhibited autoregulation of nodule number on the root half receiving delayed inoculation, regardless of plant age at the time of primary inoculation. Total nodule number plant^?1 invariably increased with later inoculation times. In contrast. NOD1 - 3 exhibited little, if any, autoregulation of nodule number. It was concluded that although Williams 82 exhibits autoregulation of nodule number and NODI - 3 does not, there was no finite limit to nodule number in either line since any delay in inoculation resulted in formation of a greater nodule number on both lines if root growth was not restricted. Nodule number in Williams 82 and NODI - 3 appears to be a function of infection sites (root size) at the time of inoculation and of subsequent plant growth.