Phylogenetic relationships among Frankia genomic species determined by use of amplified 16S rDNA sequences

Actinomycetes of the genus Frankia establish a nitrogen-fixing symbiosis with a large number of woody dicotyledonous plants. Hundreds of strains isolated from various actinorhizal plants growing in different geographical areas have recently been classified into at least nine genomic species by use of the DNA-DNA hybridization technique (M.P. Fernandez, H. Meugnier, P.A.D. Grimont, and R. Bardin, Int. J. Syst. Bacteriol. 39:424-429, 1989). A protocol based on the amplification and sequencing of 16S ribosomal DNA segments was used to classify and estimate the phylogenetic relationships among eight different genomic species. A good correlation was established between the grouping of strains according to their 16S ribosomal DNA sequence homology and that based on total DNA homology, since most genomic species could be characterized by a specific sequence. The phylogenetic tree showed that strains belonging to the Alnus infectivity group are closely related to strains belonging to the Casuarina infectivity group and that strains of these two infectivity groups are well separated from strains of the Elaeagnus infectivity group, which also includes atypical strains isolated from the Casuarina group. This phylogenetic analysis was also very efficient for classifying previously unclassified pure cultures or unisolatable strains by using total DNA extracted directly from nodules.     

Geographic distribution and genetic diversity of Ceanothus-infective Frankia strains

Little is known about Ceanothus-infective Frankia strains because no Frankia strains that can reinfect the host plants have been isolated from Ceonothus spp. Therefore, we studied the diversity of the Ceonothus-infective Frankia strains by using molecular techniques. Frankia strains inhabiting root nodules of nine Ceanothus species were characterized. The Ceanothus species used represent the taxonomic diversity and geographic range of the genus; therefore, the breadth of the diversity of Frankia strains that infect Ceanothus spp. was studied. DNA was amplified directly from nodular material by using the PCR. The amplified region included the 3' end of the 16S rRNA gene, the intergenic spacer, and a large portion of the 23S rRNA gene. A series of restriction enzyme digestions of the PCR product allowed us to identify PCR-restriction fragment length polymorphism (RFLP) groups among the Ceanothus-infective Frankia strains tested. Twelve different enzymes were used, which resulted in four different PCR-RFLP groups. The groups did not follow the taxonomic lines of the Ceanothus host species. Instead, the Frankia strains present were related to the sample collection locales.     

Diversity and specificity of Frankia strains in nodules of sympatric Myrica gale, Alnus incana, and Shepherdia canadensis determined by rrs gene polymorphism

The identity of Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct from Frankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from two M. gale nodules at a second site in Wisconsin. Frankia strains from nodules of S. canadensis belonged to a divergent subset of a cluster of Elaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankia populations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankia populations found in Myrica nodules.     

Genetic heterogeneity among Frankia isolates from root nodules of individual actinorhizal plants

Abstract Genetic variations among selected Frankia isolates from nitrogen-fixing root nodules harvested from an individual actinorhizal plant ( Elaeagnus angustifolia L. or Shepherdia argentea Nutt.) were estimated by restriction fragment analysis of their total genomic DNA. The presence of plasmids and their restriction enzyme patterns were used as additional criteria. Certain isolates from separate nodules on the same plant were found indistinguishable, being probably clones of the same strain. An endophytic passage of a strain isolated from S. argentea on another host plant, Hippophaë rhamnoides L., did not modify the structural characteristics of the genome in the reisolates obtained. However, in some cases, especially when restriction endonucleases cleaving Frankia DNA into relatively small fragments were used, multiple infection of the actinorhizal plants with different Frankia strains and the presence of more than one strain in a nodule were demonstrated. Some aspects of variability in natural populations of Frankia are discussed.     

Recent advances in the molecular analysis of inherited disease

Many important human genes have been cloned during the last ten years. In some cases, using reverse genetic techniques [Orkin, S. H. (1986) Cell 47, 845-850], disease-causing genes have been isolated whose product was previously unknown. Important examples include the dystrophin protein which, when mutated, gives rise to either Duchenne or Becker muscular dystrophy [Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P., Feener, C. and Kunkel, L. M. (1987) Cell 50, 509-517; Monaco, A. P., Bertelson, C. J., Liechti-Gallati, S. & Kunkel, L. M. (1988) Genomics 2, 90-95; Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228] and the cystic fibrosis transmembrane conductance regulator (CFTR) [Riordan, J. R., Rommens, J. M., Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Ianuzzi, M. C., Collins, F. S. & Tsui, L.-C. (1989) Science 245, 1066-1073]. Recently the technology for systematically detecting single base-pair changes by chemical methods, enzymatic methods or direct DNA sequencing has greatly expanded and simplified. In addition to providing structural information about these clinically important genes and information on disease-causing mutations, these studies have led to an increased understanding of mechanisms of mutation, to the discovery of novel genetic mechanisms and to important clinical applications of carrier detection and pre-natal diagnosis. The recent rapid progress has been made possible by the development of DNA amplification using the polymerase chain reaction (pcr) invented by Saiki and colleagues [Saiki, R. K., Chang, C-A., Levenson, C. H., Warren, T. C., Boehm, C. D., Kazazian, H. H. & Ehrlich, H. A. (1988) N. Engl. J. Med. 319, 537-541].     

Genetic diversity among Frankia strains nodulating members of the family Casuarinaceae in Australia revealed by PCR and restriction fragment length polymorphism analysis with crushed root nodules.

DNA extracted directly from nodules was used to assess the genetic diversity of Frankia strains symbiotically associated with two species of the genus Casuarina and two of the genus Allocasuarina naturally occurring in northeastern Australia. DNA from field-collected nodules or extracted from reference cultures of Casuarina-infective Frankia strains was used as the template in PCRs with primers targeting two DNA regions, one in the ribosomal operon and the other in the nif operon. PCR products were then analyzed by using a set of restriction endonucleases. Five distinct genetic groups were recognized on the basis of these restriction patterns. These groups were consistently associated with the host species from which the nodules originated. All isolated reference strains had similar patterns and were assigned to group 1 along with six of the eight unisolated Frankia strains from Casuarina equisetifolia in Australia. Group 2 consisted of two unisolated Frankia strains from C. equisetifolia, whereas groups 3 to 5 comprised all unisolated strains from Casuarina cunninghamiana, Allocasuarina torulosa, and Allocasuarina littoralis, respectively. These results demonstrate that, contrary to the results of previous molecular studies of isolated strains, there is genetic diversity among Frankia strains that infect members of the family Casuarinacaeae. The apparent high homogeneity of Frankia strains in these previous studies probably relates to the single host species from which the strains were obtained and the origin of these strains from areas outside the natural geographic range of members of the family Casuarinaceae, where genetic diversity could be lower than in Australia.     

福建省放线菌结瘤植物共生菌Frankia遗传多样性研究

[目的]了解福建省放线菌结瘤植物共生固氮菌Frankia的遗传多样性.[方法]利用16S-23SrDNA间隔区(rrn)和nifD-K基因间隔区的PCR扩增和RFLP技术,分析了福建省木麻黄、杨梅、桤木、胡颓子等共生Frankia纯培养菌株的遗传差异.[结果]17个菌株获得rrn扩增片段,2个杨梅菌株和1个胡颓子菌株扩增未成功,酶切图谱经聚类分析表明6个地点的细枝木麻黄、短枝木麻黄、粗枝木麻黄12个共生Frankia菌株同源性高,属于一个类群,2个地点的4个杨梅菌株和1个四川桤木菌株亲缘关系近,为另一类群.25个Frankia菌株的,nifD-K基因间隔区PCR-RFLP分析结果显示,7个地点的3种木麻黄14个菌株聚类为一个类群,4个地点的7个杨梅菌株、2个地点的2个四川桤木菌株以及1个台湾桤木菌株聚类为另一个类群,胡颓子菌株则为独立的类群.[结论]研究结果表明福建省共生Frankia遗传多样性丰富.     

Identification of a uniquely immunodominant, cross-reacting site in the human immunodeficiency virus endonuclease protein.

One of the features of the life cycle of retroviruses is insertion of the proviral DNA into host chromosomes. A protein encoded by the 3' end of the pol gene of the virus genome has been shown to possess endonuclease activity (D. P. Grandgenett, A. C. Vora, and R. D. Schiff, Virology 89:119-132, 1978), which is necessary for DNA integration. Sera from the majority of human immunodeficiency virus (HIV)-infected individuals react with endonuclease protein p31 in serological tests (J. S. Allan, J. E. Coligan, T.-H. Lee, F. Barin, P. J. Kanki, S. M'Boup, M. F. McLane, J. E. Groopman, and M. Essex, Blood 69:331-333, 1987; E. F. Lillehoj, F. H. R. Salazar, R. J. Mervis, M. G. Raum, H. W. Chan, N. Ahmad, and S. Venkatesan, J. Virol. 62:3053-3058, 1988; K. S. Steimer, K. W. Higgins, M. A. Powers, J. C. Stephans, A. Gyenes, G. George-Nascimento, P. A. Liciw, P. J. Barr, R. A. Hallewell, and R. Sanchez-Pescador, J. Virol. 58:9-16, 1986). It is not known, however, which part of the protein represents the target(s) for antibody response. To study this, we synthesized peptides and used them in an enzyme-linked immunosorbent assay system to map the reactivity of human immunodeficiency virus type 1 (HIV-1) antibody-positive sera to the different regions of the HIV endonuclease. A uniquely antigenic, HIV-1- and HIV-2-cross-reacting site was identified in the central part of this protein from Phe-663 to Trp-670.     

Book reviews

Book reviewed in this article:
A nti -T uberculous D rugs . H andbook of E xperimental P harmacology , Volume 84 (1988). Edited by K. Bartmann.
B acterial E ndotoxins : P athophysiological E ffects , C linical S ignificance and P harmacological C ontrol (1988). Edited by J. Levin, H.R. Buller, J.W. ten Cate, S.J.H. van Deventer & A. Sturk.
A L aboratory M anual for L egionella (1988). Edited by T.G. Harrison & A.G. Taylor.
M arine M icrobiology (1988). By B. Austin.
B acterial E nergy T ransduction (1988). Edited by C. Anthony.
S ingle C ell O il (1988). Edited by R.S. Moreton.
D iagnostic T echniques in M edical P arasitology (1988). By S.L. Fleck & A.H. Moody.     

Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina

Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated. Acetylene reduction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickel-depleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 μ mol H₂ (mg protein)⁻¹ h⁻¹ after addition of up to 1.0 μ M Ni. It is likely that the hydrogenase activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia living in symbiosis with Casuarina spp. show hydrogenase activity. Therefore, the results also indicate that the hydrogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina ., i.e., Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.     

Effect of inoculation and leaf litter amendment on establishment of nodule-forming Frankia populations in soil

High-N(2)-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 [(15)N]NO(3) 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 N(2) 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 N(2) 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 N(2) fixation rates by (15)N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N(2)-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 N(2)-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.     

Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.     

Books

Book reviewed in this article:
A ndrle , R.F. & C arroll , J.R. (eds) 1988. The Atlas of Breeding Birds in New York State.
B ergerud , A.T. & G ratson , M.W. (eds) 1988. Adaptive Strategies and Population Ecology of Northern Grouse.
C ooper , M.E. 1987. An Introduction to Animal Law.
G oriup , P.D. (ed.) 1988. Ecology and Conservation of Grassland Birds.
H udson , P.J. & R ands , M.R. 1988. Ecology and Management of Gamebirds.
J anssen , R.B. 1987. Birds in Minnesota.
J ones , P.H. 1988. The Natural History of Bardsey.
K ondratiev , A. YA. (ed.) 1988. Bulletin of the Working Group on Waders.
L ardelli , R. 1988. Atlante degli Uccelli Nidificanti net Mendrisiotto.
M arle , J.G. van & V oous , K.H. 1988. The Birds of Sumatra. B.O.U. Check-list No. 10.
P almer , R.S. (ed.) 1988. Handbook of North American Birds; Volumes 4 & 5: Diurnal Raptors.
R ose , M.R. 1987. Quantitative Ecological Theory: an Introduction to Basic Models.
S chleidt , V.M. (ed.) 1988. Der Kreis urn Konrad Lorenz.
S ick , H. 1985. Ornitologia Brasileira. 3rd edition. Vols I and II.
S ummers -S mith , J.D. 1988. The Sparrows.
S iokhin , V.D., C hernichko , I.I., A rdamatskaya , T.B. et al. 1988. Colonial Waterbirds in the Southern Ukraine: Charadriiformes.
T emple , S.A. & C ary , J.R. 1987. Wisconsin Birds: A Seasonal and Geographical Guide.
T rounson , D. & T rounson , M. 1987. Australia Land of Birds.
W oods , R.W. 1988. Guide to Birds of the Falkland Islands.     

Identification of atypical Frankia strains by fatty acid analysis

Abstract: Ineffective, non-infective actinomycetous isolates obtained from actinorhizal nodules of Coriaria nepalensis and Datisca cannabina were identified as Frankia using whole cell fatty acid analysis. The isolates exhibited fatty-acid patterns very similar to those of confirmed Frankia strains from other host plants ( Alnus, Casuarina, Colletia, Comptonia, Elaeagnus and Hippophae ). All Frankia strains, including Coriaria and Datisca isolates, showed fatty-acid profiles very distinct from those of other actinomycetes used as controls ( Actinomyces, Geodermatophilus, Nocardia, Mycobacterium and Streptomyces ). For the genus Frankia , a characteristic pattern of five fatty acids (15:0; 15:1; 16:0 iso; 17:0 and 17:1) was found. These fatty acids comprised 75% or more of the total content. All Frankia strains could be placed into three subgroups. Coriaria isolates were found in the largest subgroup which contained most Frankia strains from other hosts while ineffective strains from Alnus, Elaeagnus and Datisca were distributed in all three subgroups of Frankia .     

Sequence analysis of rpoB and rpoD gene fragments reveals the phylogenetic diversity of actinobacteria of genus Frankia

Partial rpoD, rpoB, and 16S rRNA gene sequences were obtained from databases and (or) amplified from 12 strains of Frankia. These strains belonged to either Cluster 1 (Alnus-, Myrica-, Comptonia-, and Casuarina-infective strains) or Cluster 3 (Elaeagnus-infective strain). An rpoD gene-based PCR approach was designed to allow the detection of frankiae in complex samples. Additionally, partial gene sequences obtained using 2 rpoB gene primer sets (named rpoB-1 and rpoB-2) were used to generate phylogenetic eurograms to find a molecular tool able to assess biodiversity among Frankia strains. The rpoB-2 primer set allowed separation of closely related strains and groupings representative of host plant compatibility groups. One exception to this was for strains ACN10a and ACN14a, isolated from the same geographical location. Results obtained showed that rpoB-2 is a tool of great interest to evaluate relatedness of Frankia strains, and assess biodiversity in this genus. Additionally, since rpoB-2 phylogenetic profiles of the Frankia strains studied reflected the species of host plants they were isolated from, the study of rpoB (a house-keeping gene) shows promise for future ecological studies on these symbioses.     

Book reviews

Book reviewed in this article:
P arasitology in F ocus : F acts and T rends (1988). Edited by H. Mehlman.
C olour A tlas for the D iagnosis of B acterial P athogens in A nimals (1988). Edited by W. Bisping & G. Amtsberg.
R ole of G ut F lora in T oxicity and C ancer (1988). By I.R. Rowland.
A naerobes T oday (1988). Edited by J.M. Hardie & S.P. Bordello.
T he R elease of G enetically -E ngineered M icro -O rganisms (1988). Edited by M. Sussman, C.H. Collins, F.A. Skinner & D.E. Stewart-Tull.
M icrobial T echnology in the D eveloping W orld (1988). Edited by E.J. Dasilva, Y.R. Dommergues, E.J. Nyns & C. Ratledge.
M icrobial L ipids Volume 1 (1988). Edited by C. Ratledge & S.G. Wilkinson.     

Endophyte sporulation in root nodules of actinorhizal plants

All strains of isolated Frankia possess the genetic capacity to form sporangia since, when grown in vitro, they usually sporulate freely, depending on the physical and chemical environment in which they are cultured. Endophytic sporulation involving Frankia differentiation of sporangia within root nodules has been described in only 16 host species in 9 genera within six families of actinorhizal plants. From studies published to date, endophytic sporulation cannot be correlated with specific environmental conditions surrounding the host plants. Based on the literature and on previously unpublished observations from field and greenhouse studies, an account is given of the occurrence of sporulation in actinorhizal plants with emphasis on Alnus, Casuarina, Comptonia, Elaeagnus and Myrica . The possible role of the host plant in controlling Frankia sporulation as contrasted to the control exerted by the genetic constitution of the microbial symbiont is explored.     

Co-evolution between Frankia populations and host plants in the family Casuarinaceae and consequent patterns of global dispersal

Symbioses between the root nodule-forming, nitrogen-fixing actinomycete Frankia and its angiospermous host plants are important in the nitrogen economies of numerous terrestrial ecosystems. Molecular characterization of Frankia strains using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses of the 16S rRNA-ITS gene and of the nifD-nifK spacer was conducted directly on root nodules collected worldwide from Casuarina and Allocasuarina trees. In their native habitats in Australia, host species contained seven distinctive sets of Frankia in seven different molecular phylogenetic groups. Where Casuarina and Allocasuarina trees are newly planted outside Australia, they do not normally nodulate unless Frankia is introduced with the host seedling. Nodules from Casuarina trees introduced outside Australia over the last two centuries were found to contain Frankia from only one of the seven phylogenetic groups associated with the host genus Casuarina in Australia. The phylogenetic group of Frankia found in Casuarina and Allocasuarina trees introduced outside Australia is the only group that has yielded isolates in pure culture, suggesting a greater ability to survive independently of a host. Furthermore, the Frankia species in this group are able to nodulate a wider range of host species than those in the other six groups. In baiting studies, Casuarina spp. are compatible with more Frankia microsymbiont groups than Allocasuarina host spp. adapted to drier soil conditions, and C. equisetifolia has broader microsymbiont compatibility than other Casuarina spp. Some Frankia associated with the nodular rhizosphere and rhizoplan, but not with the nodular tissue, of Australian hosts were able to nodulate cosmopolitan Myrica plants that have broad microsymbiont compatibility and, hence, are a potential host of Casuarinaceae-infective Frankia outside the hosts' native range. The results are consistent with the idea that Frankia symbiotic promiscuity and ease of isolation on organic substrates, suggesting saprophytic potential, are associated with increased microsymbiont ability to disperse and adapt to diverse new environments, and that both genetics and environment determine a host's nodular microsymbiont.     

Divergence in symbiotic interactions between same genotypic PCR–RFLP Frankia strains and different Casuarinaceae species under natural conditions

The symbiotic interactions between Frankia strains and their associated plants from the Casuarinaceae under controlled conditions are well documented but little is known about these interactions under natural conditions. We explored the symbiotic interactions between eight genotypically characterized Frankia strains and five Casuarinaceae species in long-term field trials. Characterization of strains was performed using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) for the nifD – nifK intergenic transcribed spacer (ITS) and 16S–23S ITS. Assessments of the symbiotic interactions were based on nodulation patterns using nodule dry weight and viability, and on actual N₂ fixation using the δ¹⁵N method. The PCR–RFLP patterns showed that the analyzed strains belonged to the same genotypic group (CeD group), regardless of the host species and environment of origin. The nodule viability index is introduced as a new tool to measure the viability of perennial nodules and to predict their effectiveness. The host Casuarinaceae species was a key factor influencing both the actual N₂-fixing activity of the associated Frankia strain and the viability of nodules within a location. This is the first study providing information on the symbiotic interactions between genotypically characterized Frankia strains and actinorhizal plants under natural conditions. The results revealed a way to improve a long-term management of the Casuarinaceae symbiosis.