Effect of carbon source on growth,nitrogenase and uptake hydrogenase activities of Frankia isolates from Casuarina sp.

The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied. In addition, regulation of nitrogenase and uptake hydrogenase activity by carbon sources was investigated. For each of the three isolates, JCT287, KB5 and HFPCcI3, growth was greatest on the carbon sources pyruvate and propionate. In general the carbon sources which gave the greatest growth gave the highest levels of nitrogenase activity, but repressed the activity of uptake hydrogenase. The regulation of growth, uptake hydrogenase activity and nitrogenase activity is discussed.     

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.     

Occurrence of Myrica-nodulating Frankia in Hawaiian volcanic soils

The ability of Hawaiian volcanic soils to nodulate actinorhizal Myrica cerifera, Casuarina equisetifolia, and Alnus glutinosa was determined using a host-plant bioassay. Myrica-nodulating Frankia occurred in five volcanic deposits with depositional ages ranging from 20 to 162 years before present. The oldest deposit had a mean estimated nodulation capacity from 450 to 1200 times greater than those of the younger deposits. Only the oldest deposit had high moisture content, high organic matter content, and increased vegetative cover, including an abundance of actinorhizal M. faya. Casuarina- and Alnus-nodulating Frankia were not detected in any of these volcanic deposits.     

Dinitrogen fixation in a water-stressed Alnus clone is limited by host xerotolerance

The osmotolerance, rather than the halotolerance, of the endosymbiont predicted the xerotolerance of acetylene reduction by Alnus nodulated withFrankia ARgP5 ^AG . Cloned plants ofAlnus glutinosa (L.) Gaertn. AG8022-16 were subjected to water stress under controlled conditions in an environmental growth chamber. Transpiration, stomatal conductance, and leaf water potential had decreased after successive 10 day periods of moderate (75% of water demand) and severe (50% of water demand) water stress. After severe stress had wilted the plants, reducing leaf water potential to –2.10 MPa, nitrogenase activity had fallen to 2.51 M per plant per hour. The reported rapid turnover of nitrogenase implies thatFrankia mycelium was metabolically active at this low water potential, a water potential at which no Alnus-derivedFrankia has been reported active. Although ARgP5 ^AG was similar to other such strains in halotolerance (lower limitca.–1.25 MPa), the low water potential limit for growth with glucose (a non-assimilated osmoticum) wasca.–2.53 MPa. Nitrogenase activity was apparently more limited by host xerotolerance than by endophyte xerotolerance.Journal article J-5400 of the Oklahoma Agriculture Experiment Station, Oklahoma State University, Stillwater, OK 74078, USA.     

The occurrence of Frankia in tropical forest soils of Costa Rica

Infective and effective Frankia were shown to occur in five diverse tropical forest soils of Costa Rica. Results of a plant infection assay indicated that Frankia is a common component of the soil biota in low and high elevation, primary and secondary forest soils. This is the first report of Frankia in lowland tropical rainforests of the Americas. These results suggest either a nonsymbiotic population of soil Frankia, the presence of unknown actinorhizal host species, or an ability of Frankia to be dispersed over long distances.     

A comparison of two methods and different media for isolatingFrankia from Casuarina root nodules

Four species of Casuarina were raised in the glasshouse and inoculated with nodules collected from nine different geographical areas within Australia. Isolations ofFrankia were attempted from 10 of the Casuarina-Frankia nodule combinations using two methods, a nodule dissection and a filtration method. With both techniquesFrankia isolates were obtained from four of the 10Frankia sources. Spores were not observed in sections of nodules from the four sources from whichFrankia was isolated, whereas spores were observed in the remaining six nodule sources. For selected nodule sources a range of isolation media were tried, but no improvement in the isolation success rate was achieved. The effect of host species on ease of isolation was studied. The results obtained suggested it was theFrankia strain and not the host plant species which determined the ease of isolation from Casuarina nodules.     

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.     

Alnus rubra nodulation capacity of soil under five species from harvested forest sites in coastal British Columbia

Nodulation of Alnus rubra seedlings after inoculation with soil from under A. rubra, Betula papyrifera. Rubus lacianutus, R. spectabilis, and R.ursinus on 2 recently harvested sites was compared. Nodulation capacity was low compared to other published reports, ranging from 0 to 18.9 infective units cm^-3 of soil and was significantly affected by the site and plant species. Nodulation capacity of soil under alder was significantly higher than under all other species except R. spectabilis, regardless of site. The lowest nodulation capacity was found in soil under B. papyrifera.Joint appointment with Dept. of Soil Science, Faculty of Agricultural Sciences     

Genetic Diversity of Frankia Microsymbionts in Root Nodules from Colletia hystrix (Clos.) Plants by Sampling at a Small-Scale

Summary The genetic diversity of microsymbiont Frankia from Colletia hystrix (Clos.) plants growing in a restricted area, were investigated by PCR-restriction fragment length polymorphism (RFLP) technique. DNA from field-collected nodules was amplified by PCR with primers targeting two genomic regions; one included a large portion of the 3′ end of the 16S rRNA gene, the intergenic spacer, and the 5′ end of the 23S rRNA gene and the other in the nifD–nifK intergenic region in the nif operon as a means to estimate molecular diversity. A HaeIII digestion of the PCR product allowed us to identify PCR-RFLP groups or haplotypes among the Colletia-infective Frankia strains tested. An exhaustive small-scale sampling permitted us to detect haplotypes with a low frequency in the microsymbiont population and showed that Frankia microsymbionts have a higher genetic diversity than previously reported. Fifteen haplotypes were recognized on the basis of combining the restriction patterns in each region analyzed. The haplotype designated as A3 was found with a high frequency in the five microsymbiont Frankia groups studied indicating a dominant haplotype. This haplotype was also exhibited by strain ChI4, which was isolated in 1991 in the same locality suggesting that it is the most common haplotype in this area and very stable over time.     

Infectivity and effectivity of Frankia strains from the Rhamnaceae family on different actinorhizal plants

Ten strains of Frankia isolated from root nodules of plant species from five genera of the host family Rhamnaceae were assayed in cross inoculation assays. They were tested on host plants belonging to four actinorhizal families: Trevoa trinervis (Rhamnaceae), Elaeagnus angustifolia (Elaeagnaceae), Alnus glutinosa (Betulaceae) and Casuarina cunninghamiana (Casuarinaceae). All Frankia strains from the Rhamnaceae were able to infect and nodulate both T. trinervis and E. angustifolia. Strain ChI4 isolated from Colletia hystrix was also infective on Alnus glutinosa. All nodules showed a positive acetylene reduction indicating that the microsymbionts used as inoculants were effective in nitrogen fixation. The results suggest that Frankia strains from Rhamnaceae belong to the Elaeagnus-infective subdivision of the genus Frankia.     

Effect of substrate nitrogen on the performance ofin vitro propagatedAlnus glutinosa clones inoculated with Sp+ and Sp− Frankia strains

The addition of combined nitrogen to substrate at an appropriate rate can stimulate N₂-fixation thus inreasing the efficiency of the Alnus-Frankia symbiosis. To examine how nitrogen additions can effect the peformance of different pairs of symbionts, growth and time course of N₂-fixation were studied in plants supplied with NH₄NO₃. Two cloned ofAlnus glutinosa (L.) Gaertn., propagatedin vitro, were inoculated with two strains ofFrankia (AVP3d and ACN14a) and grown in a greenhouse. Calcined montmorillonite (Totfaice^R) was used as growth substrate. Six N treatments were made up of varied amounts of NH₄NO₃ supplied in one single addition shortly before inoculation. Weekly measurements of shoot height and repeated measurements of nitrogenase activity (acetylene reduction) performed on intact root systems were used to monitor the development of the symbioses. Nitrogen treatments containing from 0.10 to 0.68 mg N g⁻¹ dry substrate stimulated N₂-fixation as well as growth. The relative performance of the two clones was different according to N treatment; one clone showed a greater benefit from the nitrogen input. Our results support the recommendation that selection of symbionts according to performance should be carried out with an input of combined nitrogen. This can provide optimum conditions for the development of each pair of symbionts.     

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.     

Isolation of infective and effective Frankia strains from root nodules of Alnus acuminata (Betulaceae)

Two Frankia strains were isolated from root nodules of Alnus acuminata collected in the Tucumano-oranense forest, Argentina. Monosporal cultures were obtained by plating a spore suspension of each strain and isolating a single colony. The strains (named AacI and AacIII) showed branched mycelia with polymorphic sporangia and NIR-vesicles. They differed in their ability to use carbon sources: the AacI strain grew well on pyruvate, while the AacIII strain grew on mineral medium supplemented with glucose or, alternatively, with sucrose. The two strains were sensitive to oleandomycin, erythromycin, kanamycin, penicillin G, streptomycin and chloramphenicol at 5 μg/ml. The AcIII strain exhibited a moderate resistance to rifampicin, ampicillin and vancomycin. The nitrogenase activity in vitro of the strains was significantly higher in basal medium without nitrogen than that determined in the presence of ammonium chloride. Both strains were infective on seedlings of Alnus glutinosa, inducing an approximately similar percentage of nodulated plants (80%), although strain AacIII produced a higher number of nodules per plant (≤15) than strain AacI (≤6). They were also effective for nitrogen fixation in planta, determined by the acetylene reduction assay. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Number of Rhizobia and delayed inoculation influence nodulation of clovers

The relationship between numbers of rhizobia and nodulation response of legumes is of considerable practical importance. Experiments were done under controlled conditions to determine the influence of numbers of Rhizobium leguminosarum biovar. trifolii on nodulation of arrowleaf clover (Trifolium vesiculosum Savi.) and crimson clover (T. incarnatum L.). Numbers of rhizobia in excess of 1000 per seed did not substantially increase earliness of nodulation or total number of nodules formed on the taproot. Nodules, however, were formed nearer the top of the taproot as numbers of rhizobia increased to 100,000 per seed. Delayed inoculation experiments indicated that nodulation sites for these clovers only remained susceptible to infection for less than 1 day. Delaying inoculation for 4 days resulted in only a 1 to 2 day delay in nodulation for arrowleaf and crimson clovers respectively and no delay for subterranean clover (T. subterraneum L.). Apparently, larger seedlings nodulated faster.     

Preliminary characterization of an ineffective Frankia derived from a spontaneously neomycin-resistant strain

Five strains of Frankia isolated from actinorhizae of 3 alder species were cultivated with various concentrations of neomycin in the medium. For one strain, resistance to neomycin was associated with lost of effectiveness. The ineffective strain was as infective as the parent strain. Protein and isoenzymes patterns showed that the ineffective and the parent strains were quite similar. The ineffective strain differentiated vesicles inside the infected host-cells.     

Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif⁺-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10⁴ cells.     

Development and function of Pisolithus and Scleroderma ectomycorrhizas formed in vivo with Allocasuarina,Casuarina and Eucalyptus

The effect of inoculating seedlings of Eucalyptus grandis, Allocasuarina littoralis and Casuarina equisetifolia with two isolates of Pisolithus and two isolates of Scleroderma from under eucalypts was examined in a glasshouse trial. Ectomycorrhizas formed extensively on Eucalyptus (23–46% fine roots ectomycorrhizal) and Allocasuarina (18–51% fine roots ectomycorrhizal). On Casuarina, the fungi were either unable to colonize the rhizosphere (one isolate of Pisolithus), or sheathed roots, resembling ectomycorrhizas, formed on 1–2% of the fine roots. Colonization of roots by one isolate of Scleroderma resulted in the death of Casuarina seedlings. Inoculation with fungi increased shoot dry weight by up to a factor of 32 (Eucalyptus), 4 (Allocasuarina) and 3 (Casuarina). Ectomycorrhizas formed in associations with Eucalyptus and Allocasuarina had fully differentiated mantles and Hartig nets in which the host and fungal cells were linked by an extensive fibrillar matrix. Sheathed roots in Casuarina lacked a Hartig net, and the epidermis showed a hypersensitive reaction resulting in wall thickening and cell death. The sheaths are described as mantles since the density and arrangement of the hyphae in the sheaths was similar to that in mantles of the eucalypt ectomycorrhizas. The intercellular carbohydrate matrix was not produced in the Casuarina mantle in association with Pisolithus, hence the mantle was not cemented to the root. These structures differ from poorly compatible associations described previously for Pisolithus and Eucalyptus. The anatomical data indicate that ectomycorrhizal assessment based on surface morphological features may be misleading in ecological studies because compatible and incompatible associations may not be distinguishable.     

Identification of a nodD-like gene in Frankia by direct complementation of a Rhizobium nodD-mutant.

Summary Clones from aFrankia At4 gene bank were pooled into groups and mass conjugated into anodD mutant ofRhizobium leguminosarum bv.viciae by triparental matings. When peas were inoculated with the pooled transconjugants, nodulation was observed. A plasmid, pAt2GX containingFrankia DNA, was isolated from bacteria recovered from these nodules. This plasmid was shown to complement anodD mutant ofR. leguminosarum bv.viciae. Thus pAt2GX contains aFrankia gene that is functionally equivalent tonodD ofR. leguminosarum bv.viciae.     

Study of the contribution of the shoot and/or root ofAlnus sp. in the compatibility between the host and a Sp+ Frankia strain using a grafting technique

Two alder species,Alnus glutinosa (L.) Gaertn. andAlnus incana (L) Moench, were inoculated with a Sp⁺ Frankia homogenate obtained fromA. incana root nodules. This inoculum formed effective nodules on the original host plant and ineffective nodules onA. glutinosa. Grafts between the two alder species were made to determine which part of the plant is involved in this phenomenon. The results obtained indicate that the compatibility between Alnus andFrankia is restricted to the root system.