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.     

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.     

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.     

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.     

Characterization and infectivity of a spontaneous variant isolated fromFrankia sp. WEY 0131391

Summary A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the originalFrankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.     

Frankia inoculation,soil biota,and host tissue amendment influence Casuarina nodulation capacity of a tropical soil

The effects of soil biota, Frankia inoculation and tissue amendment on nodulation capacity of a soil was investigated in a factorial study using bulked soil from beneath a Casuarina cunninghamiana tree and bioassays with C. cunninghamiana seedlings as capture plants. Nodulation capacities were determined from soils incubated in sterile jars at 21 °C for 1, 7, and 28 days, after receiving all combinations of the following treatments: ± steam pasteurization, ± inoculation with Frankia isolate CjI82001, and ± amendment with different concentrations of Casuarina cladode extracts. Soil respiration within sealed containers was determined periodically during the incubation period as a measure of overall microbial activity. Soil respiration, and thus overall microbial activity, was positively correlated with increasing concentrations of Casuarina cladode extracts. The nodulation capacity of soils inoculated with Frankia strain Cj82001 decreased over time, while those of unpasteurized soils without inoculation either increased or remained unaffected. The mean nodulation capacity of unpasteurized soil inoculated with Frankia CjI82001 was two to three times greater than the sum of values for unpasteurized and inoculated pasteurized soils. Our results suggest a positive synergism between soil biota as a whole and Frankia inoculum with respect to host infection.     

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 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.     

Alnus acuminata in dual symbiosis withFrankia and two different ectomycorrhizal fungi (Alpova austroalnicola andAlpova diplophloeus) growing in soilless growth medium

In this study we investigated the capacity of Andean alder (Alnus acuminate Kunth), inoculated withFrankia and two ectomycorrhizal fungi (Alpova austroalnicola Dominguez andAlpova diplophloeus ([Zeller and Dodge] Trappe and Smith), for nodulation and growth in pots of a soilless medium that contained vermiculite or a mixture of ground basalt rock and vermiculite. The seedlings were inoculated withFrankia suspensions prepared from root nodules ofA. Acuminate, followed by inoculation with spores of either one of the twoAlpova species. The seedlings were grown in a greenhouse for 12 months. The seedlings grown in the vermiculite-based growth medium containing large (1-3 mm) basalt particles andAlpova austroalnicola or medium-sized (0.5-1 mm) basalt particles andA. Diplophloeus had the heaviest shoot and root nodule dry weights and abundant ectomycorrhizal colonization. Ectomycorrhizas formed byA. Acuminate withAlpova austroalnicola is described here for the first time. Growth ofAlnus acuminate inoculated with ectomycorrhizal fungi andFrankia in the soilless primary minerals indicates that Andean alder can alter resource supply by tapping an otherwise unavailable nutrient source.     

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.     

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.     

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.     

Effects of nickel on Frankia and its symbiosis with Alnus glutinosa (L.) Gaertn

The tolerance of nickel by Frankia in culture and in symbiosis with Alnus was determined. Yield of three Frankia strains was not affected significantly by 2.25 mM nickel when cultured in propionate medium containing hydolysed casein as nitrogen source. Yield of two strains in medium without combined nitrogen, and thus reliant on fixed nitrogen, was stimulated markedly by the same nickel concentration. Utilisation of nickel for synthesis of uptake hydrogenases is presumed to be the cause of enhanced nitrogenase activity.Although growth was reduced, treatment of 2-month-old seedlings with 0.025 mM nickel for 4 weeks did not affect nodulation significantly while nitrogenase activity was doubled. Nodulation and nitrogenase activity of seedlings receiving 0.075 mM nickel were inhibited markedly, while 0.5 mM nickel was lethal to all seedlings after 4 weeks of treatment. A few small, ineffective nodules were initiated early on some of the latter seedlings, suggesting that effects of nickel on host plant processes rather than Frankia are the primary cause of inhibition of nodulation. This interpretation is supported by the retention of substantial nitrogenase activity in 10-month-old plants 1 day after the treatment with 0.59 mM nickel, when the nickel content of roots and nodules was already maximal. No nitrogenase activity was detected after 3 days, by which time the leaves were almost completely necrotic. Over a 4 day period, most nickel was retained in the roots and nodules. Supplying histidine simultaneously at concentrations equal to, or in excess of, nickel prevented wilting and leaf necrosis, but did not increase translocation of nickel to the shoot.     

Nodulation and growth response ofAllocasuarina andCasuarina species to phosphorus fertilization

To examine how soil phosphorus status affects nitrogen fixation by the Casuarinaceae —Frankia symbiosis,Casuarina equisetifolia and two species ofAllocasuarina (A. torulosa andA. littoralis) inoculated or fertilized with KNO₃ were grown in pots in an acid soil at 4 soil phosphate levels. InoculatedC. equisetifolia nodulated well by 12 weeks after planting and the numbers and weight of nodules increased markedly with phosphorus addition. Growth ofC. equisetifolia dependent on symbiotically fixed nitrogen was more sensitive to low levels of phosphorus (30 mg kg^–1 soil) than was growth of seedings supplied with combined nitrogen; at higher levels of phosphorus, the growth response curves were similar for both nitrogen fertilized and inoculated plants. The interaction between phosphorus and nitrogen treatments (inoculated and nitrogen fertilized) demonstrated that there was a greater requirement of phosphorus for symbiotic nitrogen fixation than for plant growth when soil phosphorus was low.WithAllocasuarina species, large plant to plant variation in nodulation occurred both within pots and between replicates. This result suggests genetic variation in nodulation withinAllocasuarina species. Nodulation ofAllocasuarina species did not start until 16 weeks after planting and no growth response due toFrankia inoculation was obtained at the time of harvest. Addition of nitrogen starter is suggested to boost plant growth before the establishment of the symbiosis. Growth ofAllocasuarina species fertilized with nitrogen responded to increasing levels of phosphorus up to 90 mg P/kg soil after which it declined by 69% forA. littoralis. The decrease in shoot weight ofA. littoralis, A. torulosa, C. equisetifolia andC. cunninghamiana at high phosphorus was confirmed in a sand culture experiment, and may be atributable to phosphorus toxicity.     

Biological and Cultural Characteristics of the Effective Frankia Strain HFPCcl3 (Actinomycetales) from Casuarina cunninghamiana (Casuarinaceae)

From homogenates prepared from surface-sterilized nodules ofseedlings of Casuarina cunninghamiana grown aeroponically, astrain of Frankia designated HFPCc13 was isolated and has beengrown in pure filamentous culture in a defined synthetic nutrientmedium. Vesicle and sporangium formation can be induced by removalof combined nitrogen from the medium.Frankia strain HFPCc13nodulates young seedlings of C. cunninghamiana and C. equisetifoliawithin three weeks of inoculation with an optimum root mediumpH of 6–7 for nodulation and optimum temperature of 30–35^°C. The presence of combined nitrogen in the root mediuminhibits nodulation with NH₄⁺ more inhibitory than NO₃^–.Frankia HFPCc13 does not nodulate Allocasuarina species withinthe same family nor several other possible actinorhizal plantstested. Thus this strain is quite precise in its host specificity.The rate of acetylene reduction was greater in C. cunninghamianathan the closely related species C. equisetifolia. In neitherof these host species were vesicles observed to occur withinthe infected root nodules which had been demonstrated to beactively fixing dinitrogen. Root nodules were shown to be activein acetylene reduction over a range of O₂ concentration in thegaseous environment with an optimum at about 20 per cent O₂,the ambient P_O2 of the air. The mechanism(s) for oxygen protectionof nitrogenase within the filamentous form of Frankia withinthese nodules remains to be explained. Casuarina, Frankia, nodulation, nitrogen fixation     

Studies in tetrapartite symbioses

Alnus incana seedlings were successfully inoculated with an endomycorrhizal fungus (Glomus fasciculatus), an ectomycorrhizal fungus (Paxillus involutus) and an isolate ofFrankia (ACN1) simultaneously. The effects of the inoculation treatments on the growth performance of the seedlings were evaluated under controlled conditions.The overall growth performance of the seedlings inoculated with the three organisms was better than those inoculated withFrankia, G. fasciculatus andP. involutus individually or withFrankia+G. fasciculatus andFrankia+P. involutus combinations. The highest growth performance and mycorrhizal infection occurred when the seedlings were inoculated simultaneously withFrankia+G. fasciculatus+P. involutus.     

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.     

Effects of some pure and mixedFrankia strains on seedling growth in differentAlnus species

In greenhouse experiments plants of eightAlnus species, from various parts of the world, and from different taxonomic sections, were inoculated with threeFrankia strains in order to show any possible interaction. Mixtures in equal parts of theseFrankia strains were also tried. The growth of inoculated plants was significantly higher than of the controls, with one of the three strains being superior. Mixtures of strains generally provided higher growth than the best individual strain. No interaction betweenFrankia strains andAlnus species was detected in the young plants 60 days after inoculation. Three clones ofAlnus glutinosa were inoculated with the same pure cultures ofFrankia, without producing any interaction. Inoculation time was studied in one clone and one progeny ofAlnus glutinosa. The best results were obtained with the earlier inoculation (at sowing for the progeny and at transfer to soil for thein vitro-propagated clone). The results are discussed in terms of nursery practice and field experiments for selection in breeding programmes.     

The production and utilisation of monoclonal antibodies for identification of a Frankia strain utilised as inoculum for Casuarina equisetifolia

Monoclonal antibodies were raised against Frankia 0RS020607, a strain isolated originally by H.G. Diem from nodules of Casuarina equisetifolia from Senegal. One of these antibodies, mAb8C5, was shown by ELISA to have high, but not absolute specificity for 0RS020607. This antibody was employed to investigate the mobility and persistence of 0RS020607 in plantations of C. equisetifolia. Seedlings were inoculated in pots of sand in a forest nursery with 0RS020607, with local crushed nodule suspensions or were left uninoculated. They were planted out after 5 months in experimental plots on a moderately fertile black soil site and on a low organic, oxidised red soil site. Compared with crushed nodule inoculated seedlings or uninoculated controls, growth of seedlings at transplant was improved by inoculation with Frankia 0RS020607. However, 4 years after transplant to experimental plots, the growth of trees receiving different treatments was similar. The possibility that movement of ORS 020607 between treatment plots contributed to new nodulation and enhanced growth of uninoculated trees was tested using mAb8C5 in ELISA of Frankia mycelium, extracted from the nodules of trees of the three treatments. No significant differences in reactivity were detected between nodules from uninoculated and 0RS020607 inoculated trees at either the black or the red soil sites, showing that 0RS020607 moved between treatment plots at both sites. However, at both sites, nodules from plots of trees that were inoculated originally with local crushed nodules gave reactions in ELISA that were significantly lower than values for 0RS020607 inoculated trees, possibly due to the competitive effects for new nodulation of enhancement of the indigenous population of Frankia. Serological techniques using antibodies of high specificity against Frankia strains have value for rapid screening of field samples as a preliminary for further analysis by more discriminatory techniques based on assays of genetic polymorphisms.     

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.