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.     

Variation with soil depth,topographic position and host species in the capacity of soils from an Australian locale to nodulateCasuarina andAllocasuarina seedlings

Sandy alluvial soils in a floodplain supporting a native stand ofCasuarina cunninghamiana Miq. produced about three times as many nodulated seedlings and more than twice as many nodules per nodulated seedling on roots of baitedCasuarina spp. than did clay loam red earth soils from the adjacent valley slope. Moist and well-aerated subsurficial alluvial sands had the greatest nodulation capacity of all the soils sampled. For all topographic positions, soil samples from depths greater than 20 cm promoted 76% more nodulated Casuarina seedlings than samples from the surficial 20 cm.Seedlings of three provenances ofC. cunninghamiana, together with seedlings ofC. glauca Sieb. ex Spreng.,C. cristata F. Muell ex Miq. andC. obesa Miq. developed significantly more nodules per pot and nodules per nodulated seedling in soils from this locale than seedlings of twoCasuarina equisetifolia Forst. provenances. Seedlings of two provenances ofAllocasuarina torulosa (Ait.) L. Johnson had fewer than 1% nodulated seedlings, a significantly lower level by far than that ofCasuarina seedlings.A. torulosa provenances also had significantly fewer nodulated seedlings per pot and nodules per nodulated seedling than all Casuarina hosts excepting one poorly-nodulated provenance ofC. equisetifolia.Nodulated seedlings of allCasuarina species had the capacity to fix atmospheric N₂, as indicated by acetylene-reduction capability. The presence of yellow cladodes and low rates of acetylene reduction per plant forC. cristata Miq. suggest that this association was poorly effective.     

Phosphorus-induced micronutrient disorders in hybrid poplar

The effect of calcium on the nodulation of lucerne was studied using EGTA, a specific calcium-chelator. First, the effects of the chelator were tested on hydroponically grown plants at pH 7.0. Optimal numbers of nodules were obtained in nutrient solution containing 0.2 mM CaCl₂. When 0.4 mM EGTA was given additionally, nodulation was completely inhibited. Nodulation was restored specifically with CaCl₂, but not with MgCl₂. For studies in an acid soil (pH-H₂O 5.2), lucerne seedlings were grown in rhizotrons. 67% of the seedlings became nodulated when the soil around the seed was neutralized locally with 1.0 μmol of K₂CO₃ in drops of 12 μL volume. When native calcium was removed with 2 μmol of EGTA, nodulation was reduced to 12%. However, addition of EGTA to soil resulted in a drop of pH from 6.1 to 5.2. A phosphate buffer could also not keep soil-pH sufficiently stable. Such pH-decreases could be avoided by placing agar blocks containing 6 μmol of EGTA for three hours on freshly developed roots. This treatment reduced nodulation from 87% to 32%, with soil-pH lowering only from 6.2 to 6.0. Nodulation could be restored by adding 2 μmol of CaCl₂. The depletion of soil-calcium could depress nodule formation only during the first day after inoculation.     

Nodulation and nitrogen fixation (Acetylene reduction) in desert ironwood (Olneya tesota)

Summary Acacia greggi, Cercidium floridium, and Olneya tesota seeds were inoculated with soil from beneath mature native desert trees and grown in the greenhouse on a nitrogen free media. Olneya tesota seedlings nodulated and reduced acetylene to ethylene. Nodulation or acetylene reduction was not observed in A. greggi or C. floridium. This is the first report of nodulation and nitrogen fixation in Olneya tesota.     

Influence of soil temperature on niacin and thiamine in honey mesquite

Summary The influence of soil temperature was examined on niacin and thiamine concentration in honey mesquite (Prosopis glandulosa var.glandulosa) seedlings. The seedlings were grown in soil temperature regimes of 21, 27, and 32°C in a controlled environment growth room. Nodulation randomly occurred on the roots of the seedlings, necessitating separate analysis according to the occurrence of nodulation. Roots of nodulated seedlings from the 21°C soil temperature regime contained greater quantities of niacin and thiamine compared to root samples from seedlings grown in either 27 or 32°C regimes. Niacin concentration of non-nodulated seedlings was highest in samples from seedlings grown in the 27°C soil temperature regime and lowest in samples from seedlings grown in the 21°C regime. Thiamine concentration was the greatest from non-nodulated seedlings grown in the 27°C soil temperature regime, while the thiamine concentration of non-nodulated samples from the 32°C regime was the least. Optimal soil temperature for honey mesquite root growth appears to be about 27°C. At sub-optimal soil temperatures niacin might have limited ‘growth’ while at supra-optimal soil temperatures, thiamine might be a limiting factor. College of Agricultural Sciences Contribution No. T-9-164.     

Studies of an effective strain ofFrankia fromAllocasuarina lehmanniana of the Casuarinaceae

Summary Seedlings ofCasuarina spp. andAllocasuarina spp. were grown from seed in the greenhouse and inoculated with a nodule suspension fromC. equisetifolia. Plants ofCasuarina spp. nodulated regularly and were effective in nitrogen-fixation. Only one species ofAllocasuariona, A. lehmanniana formed root nodules. Using these plants as source of inoculum, the isolation of a newFrankia sp. HFPA11I1 (HFP022 801) was made and the strain was grown in pure culture.Frankia sp. HFPA11I1 grows well in a defined medium and shows typical morphological characteristics. In media lacking combined nitrogen, the filamentours bacterium forms terminal vesicles in abundance and differentiaties large intrahyphal or terminal sporangia containing numerous spores. This strain, used as inoculum, nodulates effectively seedlings ofC. equisietifolia andC. cunninghamiana, forming nodules with verically-growing nodule roots. Although effective in acetylene reduction, the endophyte within the nodules is filamentous and lacks veiscles. When used to inoculated seedlings ofA llocasuarina lehmanniana, Frankia sp. HFPA11I1 induces root nodules which are coralloid and lacking nodule roots. The nodules are effective in acetylene reduction and the filamentous hyphae ofFrankia within the nodule lobes lack vesicles. Effective nodulation inA. Lehmanniana depends upon environmental conditions of the seedlings and proceeds much more slowly than in Casuariana.     

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.     

N2(C2H2−C2H4) fixation in two species of Ceanothus seedlings in second year postfire chaparral

Nitrogen fixation in excised root nodules of 2-year-old, postfireCeanothus tomentosus andC. leucodermis seedlings was measured over an 8-month period using the acetylene reduction method. High levels of NO₃–N and NH₄–N present in postfire soils were limited to the upper 10 cm and did not inhibit nodulation in these deeper-rooting seedlings. Decreases in acetylene reduction activity occurred with decreased soil moisture and increased soil temperature. Nitrogen gains from these two Ceanothus shrub seedlings totalled 1.6 kg N ha^–1 yr^–1.     

Soil properties and actinorhizal vegetation influence nodulation of Alnus glutinosa and Elaeagnus angustifolia by Frankia

Nodulation (mean number of nodules per seedling) was 5 times greater for Elaeagnus angustifolia than for Alnus glutinosa overall when seedlings were grown in pots containing either an upland or an alluvial soil from central Illinois, USA. However, the upland Alfisol had 1.3 times greater nodulation capacity for A. glutinosa than for E. angustifolia. The presence of A. glutinosa trees on either soil was associated with a two-fold increase in nodulation capacity for E. angustifolia. Nodulation increases for soils under A. glutinosa were obtained for A. glutinosa seedlings in the Alfisol, but decreased nodulation for A. glutinosa seedlings occurred in the Mollisol. Greatest nodulation of E. angustifolia seedlings occurred near pH 6.6 for soil pH values ranging from 4.9 to 7.1, while greatest nodulation of A. glutinosa occurred at pH 4.9 over the same pH range. Nodulation was not affected by total soil nitrogen concentrations ranging from 0.09 to 0.20%. Mollisol pH was significantly lower under A. glutinosa trees than under E. angustifolia trees. For 4- to 8-year-old field-grown trees, A. glutinosa nodule weights were negatively correlated with soil pH, while for similar aged E. angustifolia trees nodulation in the acidic Alfisol was not detected.     

Influence of soil moisture on the nodulation of post fire seedlings of Ceanothus spp. growing in the Santa Monica Mountains of Southern California

During the dry season (early May through September of 1994), following a fall 1993 wildfire, a survey of seedling nodulation was conducted at several sites in the Santa Monica Mountains of Southern California. Seedlings of Ceanothus spinosus, C. megacarpus, C. oliganthus , and C. cuneatus were manually excavated. During this period, only 12 of the 182 seedlings excavated were nodulated, and all of the nodulated seedlings were found in the relatively moist clay soils of a stream bank. No nodules were observed on the 170 seedlings excavated from the drier sites. An irrigation experiment was established in midsummer to assess whether water stress inhibits nodulation of post-fire Ceanothus seedlings. Four plots with numerous seedlings of C. cuneatus and C. spinosus were irrigated with distilled water and monitored over a 9-week period. There was a significant increase in nodulation frequency, water potential, stomatal conductance, transpiration, shoot elongation, and photosynthetic rate of irrigated seedlings compared with adjacent controls. Although these data support the hypothesis that water stress inhibits nodulation. it is unclear whether this is because of an effect of soil moisture on the nodulation capacity of the soils (i.e. on the size and physiological state of the soil Frankia population) or to a host plant response to drought which might prevent actinorhizal root infection and/or nodule development.     

Growth and Nitrogen Fixation Activity of Soybeans in the Presence of Bean Pod Mottle Virus

Pathological and physiological responses associated with the host-microsymbiont interaction of soybeans (Glycine max‘Franklin’, ‘Dyer’, ‘Centennial’, ‘Marshall’, ‘Williams’, ‘Bedford’, and ‘Peking’), which were nodulated with Rhizobium japonicum (USDA strain 3I1b-110) and inoculated with bean pod mottle virus (BPMV) in the greenhouse, varied with the cultivar and the substrate in which the plants were grown. In plants grown in a soil mixture, the virus significantly reduced canopy growth, nodulation, total N, and ureide-N content of most cultivars but symbiotic N₂ fixation was significantly reduced only for ‘Centennial’ and ‘Peking’. In vermiculite, in which the plants depended almost exclusively on symbiotically fixed N_,2, the virus reduced canopy and nodule mass of some cultivars but these reductions were significant only for ‘Franklin’. In plants grown in this substrate, however, the virus had little effect as increases or decreases in measured components of symbiotic N metabolism were nonsignificant except for the increase in leaf ureides in ‘Marshall’. Results suggest that in either soil or vermiculite the symbiotic process in certain soybean cultivars can function at nearly a normal level despite root noduleinfection with this virus.     

PCR-amplified 16S rRNA sequence analysis to confirm nodulation of Datisca cannabina L. by the endophyte of Coriaria nepalensis Wall

Different Frankia strains and crushed nodule suspensions were tested for their ability to nodulate Coriaria nepalensis and Datisca cannabina. Datisca cannabina seedlings were nodulated effectively by both crushed nodule suspension from Coriaria nepalensis and Datisca cannabina. The origin of the endophyte in Datisca nodules induced by crushed nodules of Coriaria was confirmed by comparing partial PCR-amplified 16S rRNA sequences with those of the endophytes of both plants. Coriaria seedlings could only be nodulated by crushed nodule suspensions of Coriaria nepalensis. All pure cultures of Frankia used as a single inoculum source or in combinations with a nodule filtrate, failed to induce nodulation on Coriaria. Two atypical Frankia strains Cn3 and Cn7 isolated from Coriaria nodules showed no acetylene reduction activity and did not induce nodulation on the host seedlings.     

Effects of soil acidity on nodulation ofAlnus glutinosa and viability ofFrankia

Summary Black alder seedlings were grown from seed for 7 weeks in six soils limed to various pH levels and inoculated withFrankia in two inoculation-seeding time combinations (inoculated and seeded concurrently; inoculated then seeded 5 weeks after inoculation). Three mine soils and three non-mine soils were used. Soil pHs in the study ranged from 3.6 to 7.6. In the second inoculation-seeding time combination, a series of soil samples at each of the pH levels below 7.0 were relimed to pH 7.0 immediately prior to seeding. The purpose of the study was to examine the effects of soil acidity on the nodulation of black alder byFrankia and the viability ofFrankia in acid soils. Based on the average number of nodules established per seedling, soil pH was determined to be a significant factor affecting nodulation in the mine soils. The highest levels of nodulation occurred between soil pH 5.5 and 7.2. Below pH 5.5, nodulation was reduced. There was also evidence of decreased viability of the endophyte below pH 4.5.     

Nodulation ofMedicago sativa in solution culture

Summary The time course of increase in nodule number was observed in solution cultures of controlled composition, over periods of 5 to 7 weeks following the inoculation of 8-day-old seedlings. The plants produced successive crops of nodules two to three weeks apart. Maintaining nitrate at concentrations of 0.2 or 0.5mM greatly reduced the number of nodules in the first crop but only slightly or not at all in the larger following crops, relative to the numbers in nil-nitrate control cultures. This was not due to any decrease in the sensitivity of nodulation to inhibition by nitrate. Nodulation in the nil-nitrate controlplants was restrained following their abundant first-crop nodulation. The experiments ceased to be simple tests of effects of nitrate on rate of nodule production as soon as the first crop of nodules appeared. Subsequent nodule production was then inhibited approximately equally, by prior nodulation in the abundantly nodulated control-plants, and by nitrate in the sparsely nodulated plants in the nitrate treatment.When the effect of nitrate was tested on plants comparable in size and in number of previously established nodules, it inhibited nodule production by 70–80%, whether the plants were seedlings or month-old plants, previously nodulated or not.     

Comparisons of ectomycorrhizae on pinyon pines (Pinus edulis; Pinaceae) across extremes of soil type and herbivory

Using field and greenhouse studies, we examined the relationships among pinyon pines (Pinus edulis), their ectomycorrhizal mutualists, and their moth herbivores as a function of soil fertility. We studied two soil types—the ash and cinder soils of the San Francisco volcanic field and nearby sandy loam soils. In the field, pinyons growing in cinders suffered from reduced moisture, negative nitrogen mineralization rates, low phosphate levels, reduced growth, and high moth herbivory relative to pinyons growing in sandy loam. Pinyons growing in cinders also had twofold higher levels of ectomycorrhizal colonization than their noncinder counterparts. Similarly, in the greenhouse, seedlings grown in cinders had higher levels of ectomycorrhizal colonization and greater numbers of ectomycorrhizae than seedlings grown in sandy loam. Seedling shoot growth was significantly enhanced by ectomycorrhizae in both soils. These patterns support three conclusions. First, field and greenhouse studies demonstrated that trees growing in nutrient-poor soils had higher levels of ectomycorrhizal colonization than trees growing in better soils. Second, across soil types, variation in ectomycorrhizal colonization was better predicted by soil fertility than by herbivory. However, herbivory negatively affected ectomycorrhizae in the stressful cinder environment. Third, although mycorrhizae can be parasitic under some conditions, ectomycorrhizae had mutualistic impacts on pinyon seedlings across the environmental extremes we studied.     

Nitrogenase activity of two genotypes of Acacia mangium as affected by phosphorus nutrition

The specific nodulation, nitrogenase activity (acetylene reduction) and budgets of carbon allocation to respiration by nodulated roots were examined in two provenances of Acacia mangium Willd. grown in a glasshouse for 17 weeks to investigate the effects of soil phosphorus and genotypes of the host plant on symbiotic nitrogen fixation. Application of phosphorus (0–80 mg P kg^-1 soil) increased specific nodulation (g nodule dry weight g^-1 plant dry weight) of provenance Ma11 by two-fold and the percentage of nodulated root respiration allocated to nitrogenase by 50%, but had no effect on specific activity of nitrogenase or specific respiration coupled with nitrogenase activity. Improved phosphorus nutrition increased the specific nitrogenase activity of provenance Ma9 by 2-fold, the percentage of nodulated root respiration allocated to nitrogenase, and specific nitrogenase-linked respiration by 50%, respectively, but had no effect on the specific nodulation. The percentage of respiration coupled with nitrogenase activity in nodulated root respiration by provenance Ma9 was 60–70% higher than that in provenance Ma11, regardless of phosphorus levels applied. At the optimal level of phosphorus addition (10 mg P kg^-1 soil), provenance Ma9 had a lower dry mass than provenance Ma11. This was accompanied by a lower nodulated root respiration and a higher percentage of nodulated root respiration allocated to nitrogenase activity in provenance Ma9.     

Infectivity variation of Discaria trinervis-nodulating Frankia in Patagonian soil according to season and storage conditions

Changes in the infectious capacity of soilborne Frankia from the same site may depend on environmental conditions. To test this, we examined the effect of season of sampling, sample storage protocol and storage time. The nodulation capacity of Frankia from rhizospheric soils of Discaria trinervis (Hook et Arn.) Reiche (Rhamnaceae) growing in northwest Patagonia (Argentina) was measured using the most probable number method. Soil samples were collected seasonally and either stored moist at 4°C or air dried at room temperature for few days. Old (air-dried) soil samples were also assayed. All soils nodulated D. trinervis seedlings. Nodulation units (NU) ranged from 44 (spring, moist storage) to about 1 ml⁻¹ of soil (summer moist, and summer and autumn, air-dried storage), with intermediate values in other samples. Soils stored for 12 years, 6 months or 1 week had similar NU. Frankia NU positively correlated with soil water content ( r = 0.6, P < 0.05); therefore, it is likely that soil moisture is a relevant factor regulating soilborne Frankia nodulation ability in Patagonian soils. We suggest that Frankia can remain as spores or grow saprophytically in Patagonian soils.     

Alfalfa,Medicago sativa L., in highly weathered,acid soils

Summary The effect of lime and P application on yield (top and root weigh), nodulation, intervally collected acetylene reduction (N₂-fixation), and N and Al uptake of young alfalfa (46 days growth) were investigated in greenhouse pots containing acid Bladen or Bradson topsoils. The effect on seed germination and seedling persistence under these greenhouse conditions was also recorded.Alfalfa yield and acetylene reduction increased with lime and P additions in both soils, but, predominately, with P. There was no advantage of increasing these two parameters with liming past pH 6.0 provided P was adequate. Positive relationships (R²) existed between yield and acetylene reduction, and with both factors and root weight, nodule weight, and N uptake. Increased uptake of Al by alfalfa seedlings depressed yield, but data indicate P may block Al uptake at high soil pH. There were no treatment effects on seed germination, but P application increased plant persistence in the Bladen soil.     

Nodulation patterns of actinorhizal plants in the family rosaceae

Patterns of nodulation, growth, andFrankia — host specificity have not been well characterized for the actinorhizal genera in the family Rosaceae because of the scarcity ofFrankia isolates from these taxa. Furthermore, the few isolates available from actinorhizal Rosaceae have consistently failed to nodulate plants from the host genus. In a series of experiments, species of rosaceousDryas, Cowania, Cercocarpus, Fallugia, andPurshia were inoculated withFrankia isolates, crushedDryas actinorhizae, and neoglacial soils to ascertain whether any of these inocula would effectively induce nodulation. Neoglacial soils from Alaska and Canada nodulated not only the localDryas drummondii, but alsoCercocarpus betuloides, Cowania mexicana andPurshia tridentata from distant and ecologically diverse locales as well as nonrosaceous, actinorhizal species ofAlnus, Elaeagnus, Myrica, andShepherdia. But of eightFrankia isolates, including two fromPurshia tridentata and one fromCowania mexicana, none were able to induce nodulation onPurshia orCowania species. Globular, actinorhizae-like nodules incapable of acetylene reduction were produced onC. betuloides inoculated withFrankia isolates. Crushed nodule suspensions fromDryas drummondii nodulated rosaceousCowania, Dryas andPurshia, as well as non-rosaceousElaeagnus, Myrica, andShepherdia species. Nodules produced by inoculation ofCowania mexicana andPurshia tridentata with crushed, dried nodule suspensions fromDryas drummondii reduced acetylene to ethylene, indicating nitrogenase activity for these nodulated plants. These data suggest that a similar microsymbiont infects the actinorhizal genera in the family Rosaceae.     

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

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