Showing papers on "Frankia published in 2005"

Root-based N2-fixing symbioses: legumes, actinorhizal plants, Parasponia sp. and cycads

Abstract: In the mutualistic symbioses between legumes and rhizobia, actinorhizal plants and Frankia, Parasponia sp. and rhizobia, and cycads and cyanobacteria, the N2-fixing microsymbionts exist in specialized structures (nodules or cyanobacterial zones) within the roots of their host plants. Despite the phylogenetic diversity among both the hosts and the microsymbionts of these symbioses, certain developmental and physiological imperatives must be met for successful mutualisms. In this review, phylogenetic and ecological aspects of the four symbioses are first addressed, and then the symbioses are contrasted and compared in regard to infection and symbio-organ development, supply of carbon to the microsymbionts, regulation of O2 flux to the microsymbionts, and transfer of fixed-N to the hosts. Although similarities exist in the genetics, development, and functioning of the symbioses, it is evident that there is great diversity in many aspects of these root-based N2-fixing symbioses. Each symbiosis can be admired for the elegant means by which the host plant and microsymbiont integrate to form the mutualistic relationships so important to the functioning of the biosphere.

Non-Frankia Actinomycetes Isolated from Surface-Sterilized Roots of Casuarina equisetifolia Fix Nitrogen†

Abstract: Based on partial 16S sequences, we previously described a novel group of nonsymbiotic, acetylene reduction activity-positive actinomycetes which were isolated from surface-sterilized roots of Casuarina equisetifolia growing in Mexico. An amplified rRNA restriction analysis confirmed that these actinomycetes are distinct from Frankia, a finding substantiated by a 16S rRNA gene phylogenetic analysis of two of the Mexican isolates. Further support for these actinomycetes being separate from Frankia comes from the very low DNA-DNA homology that was found. Nevertheless, the Mexican isolates may be diazotrophs based not only on their ability to grow in N-free medium and reduce acetylene to ethylene but also on the results from 15N isotope dilution analysis and the finding that a nifH gene was PCR amplified. A comparison of the nifH sequences from the various isolates showed that they are closely related to nifH from Frankia; the similarity was 84 to 98% depending on the host specificity group. An analysis of complete 16S rRNA gene sequences demonstrated that the two strains analyzed in detail are most closely related to actinobacteria in the Thermomonosporaceae and the Micromonosporaceae.

Non-Frankia actinomycetes isolated from surface-sterilized roots of Casuarina equisetifolia fix nitrogen - eScholarship

Abstract: Based on partial 16S sequences, we previously described a novel group of nonsymbiotic, acetylene reduction activity-positive actinomycetes which were isolated from surface-sterilized roots of Casuarina equisetifolia growing in Mexico. An amplified rRNA restriction analysis confirmed that these actinomycetes are distinct from Frankia, a finding substantiated by a 16S rRNA gene phylogenetic analysis of two of the Mexican isolates. Further support for these actinomycetes being separate from Frankia comes from the very low DNA-DNA homology that was found. Nevertheless, the Mexican isolates may be diazotrophs based not only on their ability to grow in N-free medium and reduce acetylene to ethylene but also on the results from N-15 isotope dilution analysis and the finding that a nifH gene was PCR amplified. A comparison of the nifH sequences from the various isolates showed that they are closely related to nifH from Frankia; the similarity was 84 to 98% depending on the host specificity group. An analysis of complete 16S rRNA gene sequences demonstrated that the two strains analyzed in detail are most closely related to actinobacteria in the Thermomonosporaceae and the Micromonosporaceae.

Nitrogen Inhibits Nodulation and Reversibly Suppresses Nitrogen Fixation in Nodules of Alnus maritima

Abstract: ADDITIONAL INDEX WORDS. seaside alder, actinorhizal plants, Frankia, ammonium nitrate, acetylene reduction assay ABSTRACT. Symbiotic associations between Alnus maritima (Marsh.) Muhl. ex Nutt. (seaside alder) and actinomycetes in the genus Frankia Brunchorst result in root nodules in which atmospheric nitrogen (N) is fi xed. The economic and environmental benefi ts of N fi xation have led to interest in inducing root nodules during production of A. maritima. Because woody plants produced in nurseries typically are provided N fertilizer, our objectives were to determine how applied N infl uences nodulation of A. maritima and to characterize how short-term changes in root-zone N affect the function of nodules. Potted seedlings were grown in perlite that was inoculated with 30 mL of soil from the root zones of mature plants in their native habitat on the Delmarva Peninsula. Each pot was drenched once daily for 10 weeks with nutrient solution that contained ammonium nitrate at 10 concentrations from 0 to 8 mM. Plants that received no ammonium nitrate formed the most nodules, and nodulation decreased linearly as ammonium nitrate increased from 0.25 to 4 mM. Plants treated with ammonium nitrate at 4 or 8 mM formed nearly no nodules, while ammonium nitrate at 0.5 mM resulted in vigorous plants with an average nodule count of 70. In a second experiment, a population of nodulated seaside alders was established by irrigating seedlings in inoculated perlite once daily with 0.5-mM ammonium nitrate for 6 weeks. Plants then were provided ammonium nitrate at 0.5, 2, or 4 mM for 2 weeks. Acetylene-reduction assays showed suppressed nodule activity among plants provided 2- and 4-mM ammonium nitrate. Daily irrigation of those plants with N-free solution subsequently led to a rapid depletion of root-zone N and to a concomitant resurgence of nodule activity. These results demonstrate that N fertilization can be managed to promote nodulation of A. maritima and show that decreased nodule activity caused by short-term increases in root-zone N is reversible. Alnus maritima is an attractive shrub or small tree native to three small, disjunct provenances in the United States (Schrader and Graves, 2000). Like other alders, A. maritima is an actinorhizal species that forms root nodules in which Frankia fi x gaseous N (Schrader and Graves, 2000; Stibolt, 1978). Unlike most woody plants that cannot benefi t directly from atmospheric N, A. maritima with functional root nodules might perform well if planted in N- poor soils. We seek to understand how the symbiosis between A. maritima and Frankia can be established and optimized during the culture of plants in nurseries. Commercial production of plants with functional root nod- ules is important for two reasons. First, growers of nodulated A. maritima might apply less N fertilizer, which would reduce production costs and the potential for environmental damage caused by run-off of irrigation water. Second, the soils in which A. maritima is planted may not contain compatible Frankia; installation of nodulated plants would overcome this possible barrier to N fi xation in the landscape. Protocols for producing nodulated A. maritima are needed. In addition, data are needed on how N fertilization affects the N fi xation of nodulated plants, and comparisons of the growth of plants reliant on fi xed N and those supplied N fertilizer would help producers assess the practicality of reducing N fertilization. Formation of functional nodules on actinorhizal plants requires the presence of compatible Frankia in edaphic environments con- ducive to bacterial infection, nodule development, and the activity of nitrogenase. Although numerous physical and chemical factors may govern the nodulation of A. maritima and the function of its

Growth, nitrogen fixation and mineral acquisition of Alnus sieboldiana after inoculation of Frankia together with Gigaspora margarita and Pseudomonas putida

Abstract: The role of tetrapartite associations among Frankia, Gigaspora margarita (an arbuscular mycorrhizal fungus), Pseudomonas putida (rhizobacterium), and Alnus sieboldiana in growth, nitrogen fixation, and mineral acquisition of A. sieboldiana was investigated. Seedlings of A. sieboldiana were inoculated with Frankia isolated from root nodules of alder, followed by inoculation of G. margarita and P. putida, and were grown for 5 months in a greenhouse. The seedlings inoculated with Frankia and G. margarita together produced the highest biomass of shoots and root nodules. Nitrogen-fixation activity, measured by acetylene reduction assay, was observed when Frankia was inoculated. The activity, on a per-nodule gram basis, decreased after G. margarita inoculation, but on a per-plant basis there was no significant difference in the activity among inoculation treatments. The mineral content in the seedlings changed after inoculation with Frankia, but not after inoculation with P. putida and/or G. margarita. The results showed a synergistic interaction among Frankia, the mycorrhizal fungus, and the rhizobacterium on the growth of A. sieboldiana.

Comparison of four constitutive promoters for the expression of transgenes in the tropical nitrogen-fixing tree Allocasuarina verticillata

Abstract: Allocasuarina verticillata is an actinorhizal tree that lives in symbiotic association with a nitrogen fixing actinomycete called Frankia. In the search for promoters that drive strong constitutive expression in this tropical tree, we studied the organ specificity of four different constitutive promoters (CaMV 35S, e35S, e35S-4ocs and UBQ1 from Arabidopsis thaliana) in stably transformed A. verticillata plants. The s-glucuronidase (gus) gene was used as a reporter and expression studies were carried out by histochemical analyses on shoots, roots and actinorhizal nodules. While the 35S promoter was poorly expressed in the shoot apex and lateral roots, both the e35S and e35S-4ocs were found to drive high constitutive expression in the transgenic non-nodulated plants. In contrast, the UBQ1 promoter was very poorly expressed and appeared unsuitable for A. verticillata. We also showed that none of the promoters studied were active in the nodule infected cells, whatever the developmental stage studied.

The effect of Frankia and Paxillus involutus on the performance of Alnus incana subsp rugosa in mine tailings

Abstract: The purpose of this study was to determine whether symbiotic nitrogen-fixing bacteria and mycorrhizal fungi act synergistically to improve plant performance when grown on heavy metal mine tailings....

Ethylene modulates the susceptibility of the root for nodulation in actinorhizal Discaria trinervis

Abstract: Ethylene is produced by plants in response to a wide variety of environmental signals and mediates several developmental processes in higher plants. We investigated whether ethylene has a regulatory function in nodulation in the actinorhizal symbiosis between Discaria trinervis and Frankia BCU110501. Roots of axenic D. trinervis seedlings showed aberrant growth and reduced elongation rate in the presence of ethylene donors [i.e. 2-aminocyclopropane carboxylic acid (ACC) and 2-chloroethylphosphonic acid (CEPA)] in growth pouches. By contrast, inhibitors of ethylene synthesis (aminoethoxyvinylglycine, AVG) or perception (Ag + ) did not modify root growth. This indicates that the development of D. trinervis roots is sensitive to elevated ethylene levels in the absence of symbiotic Frankia. The drastic response to higher ethylene levels did not result in a systemic impairment of root nodule development. Nodulation occurred in seedlings inoculated with Frankia BCU110501 in the presence of ethylene donors or inhibitors. Overall, the ability of the seedlings to shut down nodule formation in the younger portions of the root (i.e. to autoregulate nodulation) was not significantly impaired by a modification of endogenous ethylene levels. In contrast, we detected subtle changes in the nodulation pattern of the taproots. As a result of exposing the roots to CEPA, less nodules developed in older portions of the taproot. In line with this observation, AVG or Ag + caused the opposite effect, i.e. a slight increase in nodulation of the mature regions of the taproot. These results suggest that ethylene is involved in modulating the susceptibility for nodulation of the basal portion of D. trinervis seedling roots.

Carbon cost of nitrogenase activity in Frankia-Alnus incana root nodules

Abstract: The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO2 production) and nitrogenase activity (H2 production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N2:O2, thus under N2-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N2 deprivation that occur in acetylene reduction assays and during extended Ar:O2 exposures in H2 assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O2 from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO2/H2. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO2 produced per 2e− transferred by nitrogenase to reduction of N2. The results ranged from 2.6 to 3.4 mol CO2 produced per 2e−. Carbon cost expressed as g C produced per g N reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N2-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.

Genetic diversity of Frankia microsymbionts from the relict species Myrica faya (Ait.) and Myrica rivas-martinezii (S.) in Canary Islands and Hawaii.

Abstract: In the Western Canary Islands, Myrica faya and Myrica rivas-martinezii (Myricaceae) are phylogenetically close, endemic, actinorhizal species presumed to be remnants either of the European or the African Tertiary floras. Unisolated Frankia strains from field-collected nodules on Tenerife, Gomera, and La Palma Islands were compared by their rrs gene and 16S–23S intergenic spacer (IGS) restriction patterns. To compare the genetic diversity of Frankia strains from within and outside the host’s native range, nodules of M. faya field plants were collected both in Canary Islands and in Hawaii, where this species is an exotic invasive. Myrica rivas-martinezii, endemic to the Canary Islands, was sparsely nodulated in the field. Frankia strains harbored in field-collected nodules of M. faya and M. rivas-martinezii belonged to the Elaeagnaceae strains’ genetic cluster and exhibited a high degree of diversity. Frankia genotypes were specific to each host species. In the Canary archipelago, we found no relationship between site of collection and Frankia genotype for M. faya. The only exceptions were strains from site 2 in Tenerife, a location with a geological history different from the other sites sampled. Hawaiian and Canarian M. faya strains had no genotypes in common, raising questions concerning the origin of M. faya-infective Frankia in Hawaii. Nodular strains of M. rivas-martinezii from nursery plants were genetically characterized and shown to be divergent from the strains of field-collected nodules and belong to the Alnus-Casuarina strains cluster. This suggests Myrica may have the potential to nodulate with a broader range of Frankia genotypes under artificial conditions than has been detected in field-collected nodules.

Biodiversity of hydrogenases in Frankia.

Abstract: Eighteen Frankia strains originally isolated from nine different host plants were used to study the biodiversity of hydrogenase in Frankia. In the physiological analysis, the activities of uptake hydrogenase and bidirectional hydrogenase were performed by monitoring the oxidation of hydrogen after supplying the cells with 1% hydrogen and the evolution of hydrogen using methyl viologen as an electron donor, respectively. These analyses were supported with a study of the immunological relationship between Frankia hydrogenase and other different known hydrogenases from other microorganisms. Uptake hydrogenase activity was recorded from all the Frankia strains investigated. A methyl-viologen-mediated hydrogen evolution was recorded from only four Frankia strains irrespective of the source of Frankia. From the immunological and physiological studies, we here report that there are at least three types of hydrogenases in Frankia: Ni-Fe uptake hydrogenase, hydrogen-evolving hydrogenase, and [Fe]-hydrogenase. An immunogold localization study, by cryosection technique, of the effect of nickel on the intercellular distribution of hydrogenase proteins in Frankia indicated that nickel affects the transfer of hydrogenase proteins into the membrane.

Oxygen concentration affects nodule anatomy and nitrogenase activity of Alnus maritima

Abstract: Alnus maritima is a shrub that associates with N 2 -fixing Frankia in the wetlands in which it is native. Despite low concentrations in waterlogged soils, O 2 is critical to the maintenance of this symbiosis, and Frankia -infected nodules exist on roots of plants in native stands. The objective of the present study was to determine how root-zone O 2 concentration influences N 2 fixation and the anatomy of nodules on A. maritima . Root zones of plants inoculated with soil from native stands were exposed to eight O 2 concentrations. Nitrogenase activity increased with increasing O 2 concentration. Photosynthetic rate, plant dry mass, leaf N content, and nodule fresh mass were maximal in plants maintained with 15‐25% O 2 in the root zone. Nodule counts were maximal on roots maintained at 10% and above 25% O 2 , and nodules that developed at £ 2% O 2 were < 2 mm in diameter and single-lobed. Mean total area of air spaces within nodules decreased, and mean area per space increased, with increasing O 2 concentration. Seasonal and O 2 -dependent nodule pigmentation was observed. Our data illustrate that O 2 is critical to the development of functional symbioses, and that nodules of this species, which are submersed in nature, possess mechanisms for responding to their low-O 2 environment.

Characterization of a gene locus containing squalene-hopene cyclase (shc) in Frankia alni ACN14a, and an shc homolog in Acidothermus cellulolyticus

Abstract: Hopanoid lipids are pentacyclic triterpenes that are synthesized by eubacterial prokaryotes, including the two phylogenetically-related actinobacteria Frankia and Acidothermus, and that function in protection against environmental stress. In the nitrogen-fixing Frankia, hopanoids in the vesicle envelope reduce oxygen diffusion to nitrogenase, while in the thermophile Acidothermus, hopanoids may play a role in adaptation to high temperature and low pH. A 26,335 nucleotide region of the Frankia alni genome encoding the squalene-hopene cyclase gene (she) was cloned and sequenced. Contrary to several characterized shc genes in other organisms, the Frankia shc gene was not part of a hopanoid biosynthetic cluster. Rather, the neighboring genes included several regulators, oxidoreductases, transmembrane proteins as well as multidrug transporters. A second copy of the shc gene, detected by Southern hybridization, is likely to be present in F. alni. A homologous gene was amplified and sequenced in Acidothermus cellulolyticus and found to have a sequence distance of 36.9% from that of Frankia. Within the actinobacteria, phylogenetic analysis shows a close relationship between the shc gene sequences of F. alni and A. cellulolyticus.

Occurrence of Discaria trinervis nodulating Frankia in dated sediments of glacial Andean lakes.

Abstract: Short sediment cores of four glacial Andean lakes were used to assess the occurrence of Discaria trinervis-infective Frankia by performing plant bioassays. Unperturbed sediment cores, collected at 20 to 400 m distance from shorelines were sub-sampled, lyophilized and dated by the 210 Pb and 137 Cs techniques. The change in infectious capacity of lyophilized samples was determined by comparing plants inoculated with air-dried soils from lakeshores with plants inoculated with a subsample of the respective lyophilized soil. Nodulation capacity was similar for soils treated by the two procedures. Superficial sediments of the four lakes and subsurface sediments of two of the tested lakes caused nodulation in D. trinervis, which could be mostly observed after 4 to 8 weeks after of inoculation time. The long distances between sites of core collections and lakeshores, and the water depth variation of sampling points (10 to 42 m depth), would suggest that Frankia infective propagules are transported in water. Water movements during the mixing periods would allow Frankia propagules removal from superficial sediments allowing dispersion to other shores. The estimated age of sediments indicated that Frankia could retain infectivity up to ca. 50 yrs.

Identification of the truncated hemoglobin gene in Frankia

Abstract: A PCR approach was used to identify the truncated hemoglobin (trHb) gene in Frankia. Primers were designed from the conserved regions of the actinomycete TrHb sequences. With these primers, a 202 bp amplicon was detected for several Frankia strains. Amplicons for Frankia strains EANlpec, EUNlf, CN3, and EuIlc were sequenced and found to have 69-76% DNA homology with the trHbO gene from Streptomyces coelicolor. Several important amino acid residues including residues B9F, B10Y, CD1Y, E7A, and E14F that are involved in heme coordination were identified and were unique to the actinomycete cluster of trHbO genes. Phylogenetic analysis suggested that the Frankia trHb genes were grouped based on their respective genotype and clustered closest to Mycobacterium trHb genes.

Occurrence, structure, and nitrogen-fixation of root nodules of actinorhizal Arizona alder

Abstract: Actinorhizal plants are nodulated by the symbiotic, nitrogen-fixing actinomycete Frankia. The genus Alnus in the family Betulaceae is one of the 24 genera in 8 families of angiospermous plants that are actinorhizal. Arizona alder (Alnus oblongifolia Torr.) occurs in isolated populations associated with the watersheds of Madrean Sky Islands in the Southwestern United States between 1,370 and 2,285 m in elevation. We have found root nodules on alder trees from Oak Creek Canyon in central Coconino County, Arizona, and from the Santa Catalina Mountains in southeastern and central Pima County, Arizona. We describe the occurrence of nodules at two locations at or near opposite latitudinal limits of Arizona alder’s main range. Capacity of Arizona alder nodules to fix atmospheric nitrogen is confirmed by the acetylene reduction assay and the occurrence of vesicles in infected cortical cells of nodule lobes. Nodule location on roots, nodule morphology, and cellular anatomy confirm symbiotic structure similar to that of other alder species.

Correlations between the ages of Alnus host species and the genetic diversity of associated endosymbiotic Frankia strains from nodules.

Abstract: Nodule samples were collected from four alder species: Alnus nepalensis, A. sibirica, A. tinctoria and A. mandshurica growing in different environments on Gaoligong Mountains, Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS between nifD and nifK genes was directly applied to uncultured Frankia strains in the nodules. A total of 21 restriction patterns were obtained. The Frankia population in the nodules of A. nepalensis had the highest genetic diversity among all four Frankia populations; by contrast, the population in the nodules of A. mandshurica had the lowest degree of divergence; the ones in the nodules of A. sibirica and A. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated that Frankia strains from A. sibirica and A. tinctoria had a close genetic relationship. Frankia strains from A. nepalensis might be the ancestor of Frankia strains infecting other Alnus species. From these results and the inference of the ages of Alnus host species, it is deduced that there was a co-evolution between Alnus and its microsymbiont Frankia in China.

Genetic diversity of Frankia strains isolated from root nodules of Casuarina in Israel

Abstract: Eight Frankia strains were isolated from root nodules of Casuarina cunning-hamiana trees growing in a sandy loam soil at an irrigated landscaping site on Kib-butz Naan, in Israel. Analysis of the 16S ribosomal DNA gene sequence determined the diversity of the different isolated strains, and the sequences were compared to two known Frankia strains: DSM-44251, isolated from Alnus rubra (Betulaceae family) and DSM-43829, isolated from Colletia crucita (Rhamnaceae family). The phylogenetic tree constructed, based on the 16S rDNA sequencing results, revealed that the strains isolated from Casuarina cunninghamiana had a high phylogenetic similarity to the known strain isolated from Alnus rubra, whereas the sequence ho-mology of the strain isolated from Colletia crucita was located at a distant branch of the phylogenetic tree. These results demonstrate for the first time the existence of “actinorhizal” symbiosis in Israeli soil and the relation of Israeli Frankia strains to known strains from different regions of the world.

In vitro plant regeneration of Alnus acuminata H.B.K. ssp. acuminata and its root nodulation by Frankia

Abstract: A procedure for in vitro plant regeneration of Alnus acuminata from epicotyls with cotyledonary buds was developed using different media formulations with different growth regulators and carbon sources The development of multiple buds on explants at the initiation step was obtained with MS at 1/2 strength with either 1 or 2μM of BAP but not without it Multiplication gave up to 15 elongating shoots by explant, the best medium being MS supplemented with vitamins from B5 medium, 1μM of BAP and 87mM sucrose Rooting of about 88% occurred in the medium MS with 83 mM sucrose and 1μM IBA Alnus acuminata did not developed well on WPM Roots of in vitro propagated plants were nodulated by Alnus-infective Frankia The root nodules show a typical alder root nodule anatomy and differentiation pattern and effectively fixed nitrogen Rhamnaceae-infective Frankia did not nodulate in vitro cultivated Alnus acuminata suggesting that symbiotic recognition was not altered by in vitro regeneration of the plant

Genome diversity of Frankia strains from different origins and host plants by AFLP analysis

Abstract: Twenty three selected, culturable, Frankia strains belonging to different geographical origins and host-specificity groups were clustered on the basis of their AFLP patterns. A significant diversity was found among strains. The majority of strains studied corresponding to the three host-specificity groups Ainus, Casuarina and Elaeagnus (Frankia clusters 1 and 3) fell into the cluster group B. Within this group, two composite subgroups were found, Bl consisting of Frankia clusters 1 and 3. Frankia strains nodulating the Myricaceae family were not found closely related, although they were clustered within subgroup B1, CcI3 and Ce20 strains both Casuarina microsymbionts clusters in different subgroups, A and B1, respectively. The concordance between the genospecies previously described show that the grouping may reflect the taxonomic structure of the genus Frankia.

Comparative analysis of three Frankia genomes: symbiotic interaction with actinorhizal host plants and genome plasticity

Abstract: Author(s): Lapierre, Pascal; Gogarten, J.P.; Huang, Y.; Mastronunzio, J.; Rawnsley, T.; Bassi, C.A.; Tisa, L.S.; Francino, M.P.; Lapidus, Alla; Richardson, P.; Normand, P.; Benson, D.R.

Polymorphism in Alnus based Frankia of Darjeeling.

Abstract: DNA samples extracted from the root nodules of Alnus nepalensis, collected from 10 different locations of Darjeeling hills, were used to assess the genetic diversity of Frankia. The DNA samples from the nodules of naturally growing plants were used as templates in PCR, targeting different genomic regions of Frankia, namely distal, middle and proximal parts of 16S rRNA gene and nifH-D IGS region with locus specific primers. The PCR products were digested with a number of frequent (4-base) cutter restriction endonucleases. Bands were scored as present (1) or absent (0) and the clustering was done using NTSYSpc. Distinct polymorphism was found among the nodules collected from different parts of the region and those of same geographic area. These results demonstrate that genetic diversity is indeed present among the naturally occurring Frankia of Darjeeling, India.