Variable compatibility of clonedAlnus glutinosa ecotypes against ineffectiveFrankia strains

Nodulation tests onin-vitro propagated clones ofAlnus glutinosa ecotypes (forest ecotype, pioneer ecotype) withFrankia strains originating from both ecotypes indicated differences in host-plant compatibility. Inoculated plants of the pioneer ecotype clone were not infected by strains, that were unable to fix nitrogen in pure culture. Nodulation could only be induced on the clone of the forest ecotype, but no nitrogen-fixing activity could be detected. Ultra-structural observations of the nodules by SEM and TEM indicated that ineffectivity of these strains was correlated with the lack of vesicles in the infected cells. Cells were only filled with hyphae: neither sporangia nor vesicles could be detected. In contrast, effective nodules could be obtained on both alder clones after inoculation with an effective strain, showing normal development of vesicle clusters in infected cells. In pure culture the ineffective strains produced no vesicles; sporangia were found only during early stage of growth. The results demonstrate the existence ofFrankia strains which were either non-infective or ineffective on different clones ofAlnus glutinosa.     

The improvement and utilization in forestry of nitrogen fixation by actinorhizal plants with special reference toAlnus in Scotland

Summary Alnus species are used widely in Britain for land reclamation, forestry and other purposes. Rapid juvenile growth of the AmericanAlnus rubra makes it an attractive species for planting on N-deficient soils, particularly those of low organic content. In small plot trials, this species is nodulated by indigenous soil frankiae as effectively asAlnus glutinosa. Over a three year period both species return similar amounts of N to the ecosystem, estimated at up to 10–12 kg N ha^–1. Several strains ofFrankia have been isolated from local (Lennox Forest)A. rubra nodules. These differ morphologically and in their growth on different culture media, both from each other and fromA. glutinosa nodule isolates. AllAlnus isolates, however, have a total cellular fatty acid composition qualitatively similar to some other Group B frankiae. Glasshouse tests in N free culture suggest thatA. rubra nodules formed after inoculation of seedlings with American spore (–) isolates are three times more effective in N fixation than those inoculated with LennoxA. rubra spore (+) nodule homogenates. By contrast, the early growth of seedlings inoculated with spore (–)Frankia strains suggests at best a 35% improvement in N fixing activity over seedlings inoculated with LennoxA. rubra nodule isolates. Nevertheless, this improvement in activity, together with the better performance of seedlings inoculated with isolates compared with those treated with crushed nodule preparations, suggest that it would be worthwhile commercially to inoculate nursery stock with a spore (–)Frankia strain.     

Host plant growth response to inoculation withFrankia

Optimum growth conditions and inoculation regimes were determined for severalFrankia strains isolated from both Alnus and Casuarina host plants. Growth conditions were estabilished that allowed a reduction in generation time to less than 15 hours for certain Alnus derivedFrankia. Differences in plant growth response were observed with differing inoculum levels and soil mixtures. Elite strains of Alnus derivedFrankia were isolated that elicited similar growth reponses in allAlnus species tested; however, differences were observed betweenFrankia strains and plant growth response of variousCasuarina species tested.     

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.     

Alder-Frankia interaction and alder-poplar association for biomass production

Summary Alders have an important role to play in biomass producing stands because of their N₂-fixing ability and their capacity to withstand soils having an excess of moisture. The objectives of preliminary trials were (1) to find if there is any alder-genotype xFrankia-strain interaction when the effect of inoculating the bacteria was compared to no inoculation in seed beds of different species and provenances of alder, (2) to measure the possible effect of black alders interplanted in poplars compared to pure poplar plots. Two trials were laid out to study the alder-Frankia interaction. Both produced interaction. In the first one the inoculation had a favorable effect onAlnus glutinosa at age 2 years andA. cordata at age 1 and 2 and no effect onA. rubra. In the second one the inoculation had a depressive effect at age 1 on 2 of 3 provenances ofA. rubra and no effect on 1A. rubra, 3A.glutinosa and 3A. cordata provenances.A closely spaced field trial associating one black alder provenance and the poplar clone UNAL gives no superiority of mixed plots compared to pure plots. The results suggest that the N₂-fixation of alders is not profitable to poplars at age 3 with a 1.5×2 m spacing.     

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.     

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.     

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.     

Host range ofFrankia endophytes

Summary Cross-inoculation experiments with 10 pure cultured strains and 17 host species were carried out. The 10 strains were isolated from the root nodules on actinorhizal trees ranging in 9 species, 5 genera and 4 families. The host species belong to 5 genera. The pure cultured strains fromAlnus are of strong ability to infect different species of the same genus. The seedlings inoculated with these strains are able to nodulate normally. These strains can also infect and nodulate the seedlings ofMyrica californica, but not the seedlings of Elaeagnus, Casuarina andMyrica rubra. The pure cultured strains from Elaeagnus can infect and nodulate the host species in the same genus and family with an exception ofE. viridis vardelavayi, which can be only poorly nodulated by a few strains from Elaeagnus. The strains from Elaeagnus cannot infect the seedlings of Alnus andMyrica rubra. The results presented here suggest thatFrankia endophytes can be divided into two groups: Alnus group and Elaeagnus group.     

Frankia-Alnus incana symbiosis: Effect of endophyte on nitrogen fixation and biomass production

The efficiency of different FinnishFrankia strains as symbionts onAlnus incana (L.) Moench was evaluated in inoculation experiments by measuring nitrogen fixation and biomass production. Since all available pure cultures ofFrankia are of the Sp⁻ type (sporangia not formed in nodules), but the dominant nodule endophyte ofA. incana in Finland is of the Sp⁺ type (sporangia formed in nodules), crushed nodules of thisFrankia type were included. The Sp⁻ pure cultures, whether originating fromA. incana orA. glutinosa, produced with one exception, similar biomass withA. incana. The highest biomass was produced with an American reference strain fromA. viridis crispa. Using Sp⁺ nodule homogenates fromA. incana as inoculum, the biomass production was only one third of that produced by Sp⁻ pure cultures from the same host. Hence, through selection of the endophyte it is possible to exert a considerable influence on the productivity ofAlnus incana.     

A comparison of symbiotic nitrogen fixation in Scotland inAlnus glutinosa andAlnus rubra

Summary Alnus glutinosa andAlnus rubra growing in the field in Scotland show specific nitrogenase activities of the same order of magnitude. The period of maximum potential nitrogenase activity coincides with that of maximum growth in late Spring and Summer. It is suggested that the retention of nitrogenase activity into the Autumn when growth has virtually ceased may be important as a contribution to the nitrogenous reserves of the tree.Bioassay of different Scottish soils, all collected from the locality of natural stands ofAlnus glutinosa, showed wide variation in the nodulation of seedlings, although generally a soil poor for nodulation ofAlnus glutinosa generally gave poor nodulation ofAlnus rubra. Soils of pH 4.5 to 6.5, best suited for growth and nitrogen fixation of the two species, often gave nodules showing highest specific nitrogen fixing activity. Young (2 to 3 year old) plants in glasshouse or controlled environment cabinet, inoculated withAlnus glutinosa endophyte, differed from mature field grown plants, however, sinceAlnus rubra required a much larger (up to 2.5 times) mass of root nodules to fix a unit quantity of N. Microscopic comparison of the nodules of glasshouse plants showed that the proportion of cells containing the vesicular (nitrogen fixing) form of the endophyte was only slightly lower inAlnus rubra than inAlnus glutinosa and it is suggested that the differences in specific nitrogen fixing activity between the two species may reflect some incompatibility of function of theAlnus glutinosa endophyte when in symbiosis withAlnus rubra.     

Evolutionary implications of nucleotide sequence relatedness between Alnus nepalensis and Alnus glutinosa and also between corresponding Frankia microsymbionts

Frankia DNAs were isolated directly from root nodules of Alnus nepalensis and Alnus nitida collected from various natural sites in India. For comparison, a nodule sample from Alnus glutinosa was also collected from Tuebingen, Germany. Nucleotide sequence analyses of amplified 16S–23S ITS region revealed that one of the microsymbionts from Alnus nepalensis was closely related to the microsymbiont from Alnus glutinosa. A similar exercise on the host was also carried out. It was found that one sample of Alnus nepalensis was closely related to Alnus glutinosa sequence from Europe. Since both Frankia and the host sequences studied revealed proximity between Alnus glutinosa and Alnus nepalensis, it is hypothesised that the common progenitor of all the alders first entered into an association with Frankia, and the symbiotic association has evolved since.     

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.     

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.     

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.     

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.     

Diversity of Frankia strains isolated from single nodules of Alnus glutinosa

Diversity of Frankia isolates originating from lobes of single nodules collected on Alnus glutinosa root systems has been analyzed using isozyme electrophoresis method. Analysis of isozyme patterns showed no divergence among strains isolated from the same nodule. Each nodule (among 10 assayed) was inhabited by a single Frankia strain.     

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 Inoculation and Leaf Litter Amendment on Establishment of Nodule-Forming Frankia Populations in Soil

High-N₂-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [¹⁵N]NO₃ and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N₂ fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N₂ fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% ± 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% ± 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% ± 6%) than by group IV (81% ± 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N₂ fixation rates by ¹⁵N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N₂-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N₂-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

Selection and micropropagation of nodulating and non-nodulating clones ofAlnus crispa (Ait.) Pursh

Summary 600,000 seedlings ofAlnus crispa were inoculated with a 111 mixture of theFrankia strains ACN1 ^AG , AGN1 _exo ^AG and MGP10i. After 3 successive inoculations and screenings, one individual, AC-4, was selected as non-nodulating (Nod^–) with Frankiae. This selected individual AC-4 (Nod^–) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) withFrankia werein vitro propagated. The different clones ofA. crispa in culture required different kinds and concentrations of sugar during the in vitro multiplication and rooting stages. Nodulation tests using 7Frankia strains indicated that the clone AC-4 (Nod^–) was non-nodulating with 6 of the 7Frankia strains tested. One strain,Frankia ANNI, isolated from one unique nodule produced on the mother-plant AC-4, induced 38% of the AC-4 plantlets to nodulate but with a number of nodules 10 to 20 times less than the clones AC-2 (Nod⁺) and AC-5 (Nod⁺). Morphological observations of the roots of AC-4 (Nod^–) indicated that this clone had few and abnormally short root hairs.