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.     

A comparison of cultural characteristics and infectivity ofFrankia isolates from root nodules ofCasuarina species

Summary The isolations of three new strains ofFrankia were made from root nodules ofCasuarina cunninghamiana growing aeroponically. Two strains, HFPCCI1 and HFPCcI2 isolated by Lopez are typicalFrankia strains, producing sporangia among filamentous mats in culture and, in the absence of combined nitrogen, forming vesicles and showing acetylene reduction. They are red-pigmented and, although failing to nodulateCasuarina hosts, effectively nodulatedElaeagnus andHippophae. A third strain HFPCcI3 isolated by Zhang from the same source, also a typicalFrankia, can form sporangia and vesicles in culture and reduce acetylene, is unpigmented, fails to nodulateElaeagnus but effectively nodulatesC. cunninghamiana andC. equisetifolia. Comparisons are made among all of theCasuarina isolates in our collection from around the world (twelve in all) with regard to their cultural characteristics and capacity to infect host plant species. Questions are raised about the specificity of the various isolates and their possible affinities. Opportunities are suggested for inoculation of seedlings for forestry and field application using the infective, effective strains now available.     

Influence of inoculation on yield ofAlnus glutinosa in the Netherlands

Summary The occurrence and the infectivity of Frankia, the root-nodule endophyte ofAlnus glutinosa, were studied in different kinds of soil in the Netherlands. Both field and pot experiments indicated that many soils, on which alders have not been grown before, had low numbers of endogenous Frankia or none at all. Inoculation of these soils usually enhanced growth and nodulation of alders.The effect of fertilizer treatments on growth and nodulation ofA. glutinosa were studied in experimental plots. Alders grown in sandy soils, dressed with farmyard manure had the highest yield and the most nodules. The influence of inoculation with homogenates of Sp(+) and Sp(–) nodules and with a pure culture of Frankia AvcIl were studied in pot experiments. The quantity of different kinds of inoculum needed to obtain good growth and nodulation of alder was estimated. The results indicated that addition of a nodule homogenate of 90 g fresh AvcIl Sp(+) nodules is sufficient to inoculate one hectare of nursery soil to produce 10 nodules per plant, while a thousand times larger amount of inoculum is necessary when Sp(–) nodules are used. The limitations and the potentials of using nodule homogenates and pure cultures of Frankia for inoculation in forestry are discussed.     

Morphology,physiology and infectivity of twoFrankia isolates An 1 and An 2 from root nodules ofAlnus nitida

Summary Two different strains, An 1 and An 2, were obtained from root nodules ofAlnus nitida Endl., collected from one locality in the area of its natural habitat near Bahrin, District Swat, Pakistan. The light and electron microscopy of the isolates revealed the occurrence of septate and branched hyphae bearing sporangia and vesicles. The strains differed in their growth requirements, nitrogen-fixing ability and production of extracellular pigments, thus indicating the existence of more than oneFrankia strain in the same locality. In the absence of combined nitrogen in the medium strain An 1 formed vesicles and fixed N₂ (up to 200 nmol C₂H₄. mg protein^–1.h^–1), while strain An 2 under the experimental conditions formed only few vesicles and fixed N₂ at a very low rate (ca 10 nmol C₂H₄. mg protein^–1 .h^–1). The nitrogenase activity of strain An 1 was strongly affected by the O₂ concentration.Frankia An 1 and An 2 were infective and effective onA. nitida andA. glutinosa but not onDatisca cannabina andElaeagnus umbellata. Both An 1 and An 2 strains were more infective and effective onA. glutinosa thanFrankia strains AvcIl and CpI1.     

Glasshouse evaluation of the growth ofAlnus rubra andAlnus glutinosa on peat and acid brown earth soils when inoculated with four sources ofFrankia

The effects of soil type (an acid peat and 2 acid brown earths) andFrankia source (3 spore-positive crushed nodule inocula and spore-negative crushed nodules containing the singleFrankia ArI5) on nodulation, N content and growth ofAlnus glutinosa andA. rubra were determined in a glasshouse pot experiment of two years duration. Plants on all soils required additional P for growth. Growth of both species was very poor on peat withA. glutinosa superior toA. rubra. The former species was also superior toA. rubra on an acid brown earth with low pH and low P content. Some plant-inoculum combinations were of notable effectivity on particular soils but soil type was the major source of variation in plant weight. Inoculation with crushed nodules containingFrankia ArI5 only gave poor infection of the host plant, suggesting that inoculation with locally-collected crushed nodules can be a preferred alternative to inoculation withFrankia isolates of untested effectivity. Evidence of adaptation ofFrankia to particular soils was obtained. Thus, while the growth of all strains was stimulated by mineral soil extracts, inhibitory effects of peat extracts were more apparent with isolates from nodules from mineral soils than from peat, suggesting that survival ofFrankia on peat may be improved by strain selection.     

Restriction pattern analysis of genomic DNA ofFrankia isolates

Summary Total genomic DNAs ofFrankia isolates were subjected to restriction enzyme digestion and subsequent agarose gel electrophoresis. Restriction fragment banding patterns were unique for each isolate and may therefore be used as a method to distinguish between isolates which may be morphologically indistinguishable. This method might be useful for practical purposes such as tracing specificFrankia strains during field studies.     

Observations on the ultrastructure ofFrankia sp. in root nodules ofDatisca cannabina L.

Summary The fine structures of the microsymbiont inside the root nodules ofDatisca cannabina have been studied by light, by transmission- and by scanning-electron microscopy. The endophyte is prokaryotic and actinomycetal in nature. The hyphae are septate and branched, diameter 0.3–0.5 m. The tips of hyphae are swollen to form electron-dense, clubshaped to filamentous vesicles, ranging in diameter: 0.4–1.4 m. The endophyte penetrates through walls of the cortial cells. The infected zone is kidney shaped and confined to one side of the acentric stele. The orientation of infection is reversed from other actinorhizae exceptCoriaria. The hyphae are near the host cell wall and vesicles are directed towards the central vacuole. Vesicles are aseptate and no collapsing of the vesicle cell wall (void area) has been observed. Vesicle clusters structures are globular with an opening at one side of the cluster. The host cell is multinucleate or contains a lobed nucleus. Groups of mitochondria are located in between the hyphae, suggesting a strong association between the host and the endophyte for energy supply and amino acid production. The consequences of the inability to separate the mitochondria from the vesicle clusters in nodule homogenates in physiological studies have been discussed.Isolated vesicles clusters showed dehydrogenase activity, indicated by the presence of formazan crystals, after incubation with NADH and NBT. Strongest reducing activity was found within the vesicles. The possible role of filamentous vesicles in nitrogen fixation has been discussed.     

Distribution of spore-positive and spore-negative nodules in stands ofAlnus glutinosa andAlnus incana in Finland

Summary The distribution of spore positive (Sp⁺) and spore negative (Sp⁻) nodules on the two native alder species (A. incana andA. glutinosa) in Finland was investigated. Nodules were collected throughout the country from different ecosystems (forests, swamps, lake- sea- and riversides, old pastures and fields as well as from alder plantations). OnA. incana Sp⁺ nodules predominated, whereas onA. glutinosa the vast majority of the nodules were of the Sp⁻ type. Sp⁺ nodules onA. glutinosa were found only at sites where the two alder species grew close together. This distribution pattern indicates an association of nodule type with alder species, the reasons for which are discussed. Indications of saprophytic growth in the Sp⁻ strain were also found.     

Nitrogenase activity in root nodule homogenates ofAlnus incana

Summary Root nodule homogenates of actinorhizal plants may representFrankia in a symbiotic stage but released from environmental influence of the host plant. Anaerobic homogenization with a blender in buffer supplied with sucrose, polyvinylpyrrolidone and reducing substances gave three times higher yields of nitrogenase activity (C₂H₂-reduction) than crushing the nodules in liquid nitrogen. The activity in the homogenates was very reproducible and was, on average, nearly twice as high as the activity in excised nodules and c. 10% of the activity in intact plants. The difference in activity between excised nodules and intact plants was, roughly by halves, due to removal of the root system from the pot and to excision of the nodules. The nitrogenase activity in the homogenates was slightly higher when nodule excision was done in Ar or under water as well as after treatment of the homogenate with toluene or Triton X-100 or osmotic shock. These gains in activity were considered too small to outweigh the increased complications of preparing homogenates for routine use. Due to the reproducible recovery of nitrogenase in the homogenates the technique seems useful for physiological studies on nitrogen fixation inAlnus incana.     

Growth increment ofAlnus glutinosa upon dual inoculation with effective and ineffectiveFrankia strains

Investigations on the ecological function of ineffectiveFrankia strains and their behaviour in competition with effectiveFrankia strains indicated an enhanced plant growth upon dual inoculation with increasing amounts of effective (i.e. N₂-fixing)Frankia strains and simultaneous inoculation with a constant amount of an ineffectiveFrankia strain. Enhanced plant growth was measured as increase in plant height and total dry weight at constant shoot/root ratio. The stimulating effect of the ineffective strain was independent of the plant clone and was obtained with bothAlnus glutinosa clones W I and B II, which were resistant and susceptable, respectively, to the ineffective strain. Stimulation was also independent of the nodulation conditions. Short-term studies (7 weeks) under axenic conditions and greenhouse experiments during 3 months showed comparable results, not only in plant growth but also in nodule formation. Increment in plant growth was not necessarily correlated to higher nodule formation with the effectiveFrankia strains.     

Performance ofin vitro propagatedAlnus glutinosa (L.) Gaertn. clones inoculated withFrankiae

Summary ThreeAlnus glutinosa (L.) Gaertn. clones, obtained byin vitro propagation techniques, were inoculated with four strains ofFrankia. The ability of these clones to nodulate and fix nitrogen was previously reported; this study deals with the performance of 12 different combinations of pairs of symbionts.Shoot fresh weight, shoot height and collar diameter were measured 60 and 82 days after inoculation. Shoot fresh weight seems to be more sensitive and reliable than the other parameters. Nitrogenase activity, measured by the acetylene reduction assay, was assayed 78 days after inoculation and was consistent with the biomass measurements.Better growth was observed when type N strains were used. Significant growth differences were observed between clones AG-2 and AG-8 on the one hand and clone AG-4 on the other. Thus, the use of genetically defined host plants and microsymbionts permitted the demonstration of significant performance variation even among cloned plants from the same provenance (AG-4 and AG-8).The duration of the experiment influenced the results with differences becoming less significant with time. This might be caused by an external limiting factor such as the pot size, competition for light,etc. But it could also be indicative of differences in nodulation speed among the treatments.     

Survival ofFrankia strains introduced into soil

Factors affecting the establishment of Alnus/Frankia symbioses were studied partly by following the survival ofFrankia strains exposed to different soil conditions, and partly by investigating the effect of pH on nodulation. TwoFrankia strains were used, both of the Sp⁻ type (sporangia not formed in nodules). One of the strains sporulated heavily, while the other formed mainly hyphae. The strains originated fromAlnus incana root nodules growing in soils of pH 3.5 and 5.0. The optimum pH for their growth in pure culture was found to be 6.7 and 6.2, respectively. The strains were introduced into twoFrankia-free soils, peat and fine sand. Their survival, measured as the persistance of nodulation capacity using the plant infection technique, was followed for 14 months. The survival curves of the strains were similar despite the morphological differences between the strains in pure culture. The nodulation capacities declined over time both at 14 and 22°C. Survival was better in soils limed to a pH above 6 than in soils at their original pH (peat 2.9, fine sand 4.2). The effect of pH on nodule formation in Alnus seedlings by theFrankia strains was studied in liquid culture. The number of nodules increased linearly within the pH range studied (3.5–5.8). No nodules were formed at pH 3.5.     

Characterization of genetic diversity ofFrankia strains in nodules ofAlnus nepalensis (D. Don) from the Hengduan Mountains on the basis of PCR-RFLP analysis of thenifD-nifK IGS

Nodule samples from 90A. nepalensis individuals were collected at five sites in the Hengduan Mountains. PCR-RFLP analysis of IGS betweennifD andnifK genes was directly applied to unculturedFrankia strains in the nodules. Sizes of thenifD-nifK IGS amplicons and genetic distance between the RFLP patterns from these samples were noticeably different, indicating significant genetic variation in theFrankia population. There were some nodule samples, which produced more than one PCR fragment, and compound RFLP patterns, indicating thatFrankia strains with different PCR-RFLP patterns coexisted in the same host plant under natural conditions. Among the 29 restriction patterns obtained, 5 patterns were found in more than one population and occurred in the majority of samples, while each of the other 24 patterns were represented by only one or two samples and were endemic to a particular population. From the calculatedGst and UPGMA cluster analysis, genetic diversity ofFrankia strains was inferred to be related to climate and glaciation history in the Hengduan Mountains.     

Effective nodulation ofCoriaria myrtifolia L. induced byFrankia strains fromAlnus glutinosa L. (Gaertn.)

The present contribution covers the cross-inoculation between two actinorhizae belonging to different genera and families, mainlyAlnus glutinosa andCoriaria myrtifolia. Frankia strains isolated fromA. glutinosa received from the Netherlands (LDAgp₁r₁, LDAgn₁) and from Scotland (UGL010708), induced a fully effective nodulation onC. myrtifolia. The same effect was caused by a nodule extract fromA. glutinosa. The reverse, a crushed-nodule inoculum fromC. myrtifolia nodulated all theA. glutinosa seedlings, though nodules formed were less effective than those induced by the other inocula. Re-isolation of thoseFrankia strains from the nodules formed onA. glutinosa was readily obtained, whereas attempts to re-isolate them from the nodules formed onC. myrtifolia failed, suggesting that isolation procedures different to those employed should be tried.     

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.     

Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

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.     

Growth regulators,Rhizobium and nodulation in peas

The cytokinin content of roots and nodules of pea and the culture supernatants from two strains of Rhizobium leguminosarum has been examined. Roots, nodules and wild-type Rhizobium culture medium contained very little cytokinin as indicated by bioassay. Chemical ionisation gas chromatography-mass spectrometric analysis of the isopentenyladenine content of the culture medium from the Rhizobium strains confirmed that the content of the wild-type was low (approx. 1 ng dm^-3) but that it was increased by the introduction of the Agrobacterium Ti plasmid into the Rhizobium strain.Abbreviations CI chemical ionisation - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - iPAde isopentenyladenine - MIM multiple ion monitoring     

Uptake and evolution of H2 and reduction of C2H2 by root nodules and nodule homogenates ofAlnus glutinosa

Summary In the growing season no net H₂ evolution is detected when root nodules ofAlnus glutinosa are incubated in air or in argon containing 20% O₂. Due to the hydrogenase activity, N₂-fixing root nodules consume added H₂ at a rate of about 1.4 moles H₂.g fresh nodule^–1.h^–1. The uptake of H₂ is only found in summer. At the end of the season, in autumn, nodules evolve significant quantities of H₂ although the nodules still continue to fix nitrogen. In-vitro studies with fractionated homogenates of summer-harvested nodules show that the recovery of the hydrogenase is high when using methylene-blue or phenazine metasulfate as electron acceptors. No hydrogenase activity is detected in homogenates of autumn-harvested nodules.The hydrogenase is localised in the microsymbiont.     

Distribution of spore-positive and spore-negative nodules ofAlnus incana ssp.rugosa in Maine,USA

The distribution of spore-positive (sp+) and spore-negative (sp−) root nodules ofAlnus incana ssp.rugosa (DuRoi) Clausen (speckled alder) was examined at 29 sites with a wide range of environmental conditions in Maine, USA. These included: pH 3.4 to 7.0, soil texture ranging from coarse gravel to clay to organic soils, elevation from 3 to 591 m and latitude 43 to 47°N. Habitat types included disturbed areas, streamsides, swamps and old fields. Sp (−) nodules were substantially more common, making up 76% of all nodules, whereas only 24% were sp (+). Sp (−) nodules often occurred in pure stands and predominated at disturbed sites with mineral soils at the surface and in old fields and swamps with pH>4.0 Sp (+) nodules were nearly always found in mixture with sp (−) nodules. They occurred primarily at streamside and lakeshore sites where they made up 40% of the nodules and at sites with pH<4.0 regardless of habitat type. It is suggested that sp (−) strains ofFrankia may be maintained at a site by saprophytic growth in soil and thus nodulate newly established hosts, whereas sp (+) strains may be maintained primarily by spore production within nodules and thus depend on extended presence of the host.