Recent advances in the biogeography and genecology of symbiotic Frankia and its host plants

Molecular phylogenetic approaches have begun to outline the origin, distribution and diversity of actinorhizal partners. Geographic isolation of Frankia and its host plants resulting from shifting continents and dispersal patterns have apparently led to the development of Frankia genotypes with differing affinities for host genera, even within the same plant family. Actinorhizal plant genera of widespread global distribution tend to nodulate readily even outside their native ranges. These taxa may maintain infective Frankia populations of considerable diversity on a broad scale. Arid environments seem to have distinctive actinorhizal partnerships, with smaller and more specific sets of Frankia symbionts. This has led to the hypothesis that some host families have taxa that are evolving towards narrow strain specificity, perhaps because of drier habitats where fewer Frankia strains would be able to survive. Harsh conditions such as water-saturated soils near lakes, swamps or bogs that are typically acidic and low in oxygen may similarly lessen the diversity of Frankia strains present in the soil, perhaps limiting the pool of frankiae available for infection locally and, at a larger scale, for natural selection of symbiotic partnerships with host plants. Recent molecular ecological studies have also provided examples of Frankia strain sorting by soil environment within higher order cluster groupings of Frankia host specificity. Future frontiers for ecological research on Frankia and actinorhizal plants include the soil ecosystem and the genome of Frankia and its hosts.     

Biology of Frankia strains, actinomycete symbionts of actinorhizal plants.

Frankia strains are N2-fixing actinomycetes whose isolation and cultivation were first reported in 1978. They induce N2-fixing root nodules on diverse nonleguminous (actinorhizal) plants that are important in ecological successions and in land reclamation and remediation. The genus Frankia encompasses a diverse group of soil actinomycetes that have in common the formation of multilocular sporangia, filamentous growth, and nitrogenase-containing vesicles enveloped in multilaminated lipid envelopes. The relatively constant morphology of vesicles in culture is modified by plant interactions in symbiosis to give a diverse array of vesicles shapes. Recent studies of the genetics and molecular genetics of these organisms have begun to provide new insights into higher-plant-bacterium interactions that lead to productive N2-fixing symbioses. Sufficient information about the relationship of Frankia strains to other bacteria, and to each other, is now available to warrant the creation of some species based on phenotypic and genetic criteria.     

Phylogenetic relationships among actinorhizal plants. The impact of molecular systematics and implications for the evolution of actinorhizal symbioses

A review of recent molecular systematic studies of actinorhizal plants and their Frankia endosymbionts is presented. For comparative purposes, a discussion of recent studies pertaining to the evolution of nodulation in the legume-rhizobium system is included. Molecular systematic studies have revealed that actinorhizal plants are more closely related than current taxonomic schemes imply. Broad-based analyses of the chloroplast gene rbcL indicate that all symbiotic root-nodulating higher plants belong to a single large clade. More focused molecular analyses of both legume and actinorhizal hosts within this large clade indicate that symbioses have probably arisen more than once. By comparing host phylogenies and recently published bacterial phylogenies, we consider the coevolution of bacterial symbionts with their actinorhizal hosts.     

Casuarina root exudates alter the physiology, surface properties, and plant infectivity of Frankia sp. strain CcI3

The actinomycete genus Frankia forms nitrogen-fixing symbioses with 8 different families of actinorhizal plants, representing more than 200 different species. Very little is known about the initial molecular interactions between Frankia and host plants in the rhizosphere. Root exudates are important in Rhizobium-legume symbiosis, especially for initiating Nod factor synthesis. We measured differences in Frankia physiology after exposure to host aqueous root exudates to assess their effects on actinorhizal symbioses. Casuarina cunninghamiana root exudates were collected from plants under nitrogen-sufficient and -deficient conditions and tested on Frankia sp. strain CcI3. Root exudates increased the growth yield of Frankia in the presence of a carbon source, but Frankia was unable to use the root exudates as a sole carbon or energy source. Exposure to root exudates caused hyphal "curling" in Frankia cells, suggesting a chemotrophic response or surface property change. Exposure to root exudates altered Congo red dye binding, which indicated changes in the bacterial surface properties at the fatty acid level. Fourier transform infrared spectroscopy (FTIR) confirmed fatty acid changes and revealed further carbohydrate changes. Frankia cells preexposed to C. cunninghamiana root exudates for 6 days formed nodules on the host plant significantly earlier than control cells. These data support the hypothesis of early chemical signaling between actinorhizal host plants and Frankia in the rhizosphere.     

Low genetic diversity among Frankia spp. strains nodulating sympatric populations of actinorhizal species of Rosaceae, Ceanothus (Rhamnaceae) and Datisca glomerata (Datiscaceae) west of the Sierra Nevada (California)

Frankia spp. strains typically induce N2-fixing root nodules on actinorhizal plants. The majority of host plant taxa associated with the uncultured Group 1 Frankia strains, i.e., Ceanothus of the Rhamnaceae, Datisca glomerata (Datiscaceae), and all actinorhizal members of the Rosaceae except Dryas, are found in California. A study was conducted to determine the distribution of Frankia strains among root nodules collected from both sympatric and solitary stands of hosts. Three DNA regions were examined, the 5' end of the 16S rRNA gene, the internal transcribed spacer region between the 16S and 23S rRNA genes, and a portion of the glutamine synthetase gene (glnA). The results suggest that a narrow range of Group 1 Frankia spp. strains dominate in root nodules collected over a large area of California west of the Sierra Nevada crest with no apparent host-specificity. Comparisons with Group 2 Frankia strain diversity from Alnus and Myrica within the study range suggest that the observed low diversity is peculiar to Group 1 Frankia strains only. Factors that may account for the observed lack of genetic variability and host specificity include strain dominance over a large geographical area, current environmental selection, and (or) a past evolutionary bottleneck.     

Isolation of an effective strain of Frankia from nodules of Discaria trinervis (Rhamnaceae)

Frankia strain BCU110501 was isolated from root nodules of the native Patagonian actinorhizal plant Discaria trinervis. The strain was grown on BAP medium with sodium propionate or glucose as carbon sources. Colonies grown in nitrogen-free medium showed branched hyphae bearing polymorphic sporangia and vesicles, which were capable of nitrogen fixation. Old cultures produced a red pigment. The infectivity and effectivity of a Frankia strain isolated from Discaria on its own host, D. trinervis and also in D. chacaye, is reported for the first time. Frankia BCU110501 has physiological properties that are intermediate between categories proposed by Lechevalier et al. (1983) to classify Frankia.     

Assessing the phylogeny of Frankia-actinorhizal plant nitrogen-fixing root nodule symbioses with Frankia 16S rRNA and glutamine synthetase gene sequences

Actinomycetes from the genus Frankia induce nitrogen-fixing root nodules on actinorhizal plants in the "core rosid" clade of eudicots. Reported here are nine partial Frankia 16S rRNA gene sequences including the first from host plants of the rosaceous genera Cercocarpus and Chamaebatia, 24 partial glutamine synthetase (GSI; glnA) sequences from Frankia in nodules of 17 of the 23 actinorhizal genera, and the partial glnA sequence of Acidothermus cellulolyticus. Phylogenetic analyses of combined Frankia 16S rDNA and glnA sequences indicate that infective strains belong to three major clades (I-III) and that Clade I strains consisting of unisolated symbionts from the Coriariaceae, Datiscaceae, Rosaceae, and Ceanothus of the Rhamnaceae are basal to the other clades. Clock-like mutation rates in glnA sequence alignments indicate that all three major Frankia clades diverged early during the emergence of eudicots in the Cretaceous period, and suggest that present-day symbioses are the result of an ancestral symbiosis that emerged before the divergence of extant actinorhizal plants.     

Molecular phylogenies of plants and Frankia support multiple origins of actinorhizal symbioses

Molecular phylogenetic trees were reconstructed from nucleotide sequences of nifH and 16S rDNA for Frankia and of rbcL for actinorhizal plants. Comparison of Frankia phylogenetic trees reconstructed using nifH and 16S rDNA sequences indicated that subgroupings of both trees correspond with each other in terms of plant origins of Frankia strains. The results suggested that 16S rDNAs can be utilized for coevolution analysis of actinorhizal symbioses. Frankia and plant phylogenetic trees reconstructed using 16S rDNA and rbcL sequences were compared. The comparison by tree matching and likelihood ratio tests indicated that although branching orders of both trees do not strictly correspond with each other, subgroupings of Frankia and their host plants correspond with each other in terms of symbiotic partnership. Estimated divergence times among Frankia and plant clades indicated that Frankia clades diverged more recently than plant clades. Taken together, actinorhizal symbioses originated more than three times after the four plant clades diverged.     

Symbiosis between Frankia and actinorhizal plants: root nodules of non-legumes

In actinorhizal symbioses, filamentous nitrogen-fixing soil bacteria of the genus Frankia induce the formation of nodules on the roots of a diverse group of dicotyledonous plants representing trees or woody shrubs, with one exception, Datisca glomerata. In the nodules, Frankia fixes nitrogen and exports the products to the plant cytoplasm, while being supplied with carbon sources by the host. Possibly due to the diversity of the host plants, actinorhizal nodules show considerable variability with regard to structure, oxygen protection mechanisms and physiology. Actinorhizal and legume-rhizobia symbioses are evolutionary related and share several features.     

Relationships among pure cultured strains of Frankia based on host specificity

Fifty strains of Frankia were tested for their ability to nodulate six species of actinorhizal plants. Pure cultured strains were used to inoculate seedlings of Alnus glutinosa (L.) Gaertn., Alnus rubra Bong., Casuarina equisetifolia L., Elaeagnus angustifolia L., Hippophaë rhamnoides L. and Myrica cerifera L. in nutrient solution culture. From the results of this study, host inoculation groups among the actinorhizal plants were defined. Although overlap between host inoculation groups appears to be common, the results from this study did not support the view that Frankia strains are promiscuous. All Frankia strains tested in this study could easily be classified into four major host-specificity groups.     

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.     

Early interactions between Alnus glutinosa and Frankia strain ArI3. Production and specificity of root hair deformation factor(s)

Actinomycetes from the genus Frankia are able to form symbiotic associations with more than 200 different species of woody angiosperms, so called actinorhizal plants. Many actinorhizal plants are infected via deformed root hairs. Factor(s) eliciting root hair deformation in actinorhizal symbioses have been found to be released into the culture medium, but the factor(s) has (have) not yet been characterized. In the present work, we describe the constitutive production of factor(s) by Frankia strain ArI3 causing root hair deformation on Alnus glutinosa . Deformation was detected after 4–5 h of incubation with both Frankia cultures and their cell-free culture filtrates. When culture filtrate was used, deformation was concentration dependent. A contact time of 2 min between culture filtrate and host roots was sufficient to induce subsequent root hair deformation. No root hair deformation on A. glutinosa could be detected with purified Nod factors from Rhizobium meliloti or R. leguminosarum biovar viciae . No correlation was found between Frankia strains belonging to different host specificity groups and their ability to deform root hairs on A. glutinosa. However, strains not able to deform root hairs on A. glutinosa were also unable to nodulate.     

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.     

Molecular phylogeny of the actinorhizal Hamamelidae and relationships with host promiscuity towards Frankia

Several of the most studied actinorhizal symbioses involve associations between host plants in the subclass Hamamelidae of the dicots and actinomycetes of the genus Frankia. These actinorhizal plants comprise eight genera distributed among three families of ‘higher’ Hamamelidae, the Betulaceae, Myricaceae, and Casuarinaceae. Contrasting promiscuity towards Frankia is encountered among the different actinorhizal members of these families, and a better assessment of the evolutionary history of these actinorhizal taxa could help to understand the observed contrasts and their implications for the ecology and evolution of the actinorhizal symbiosis. Complete DNA sequences of the chloroplast gene coding for the large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) were obtained from taxa representative of these families and the Fagaceae. The phylogenetic relationships among and within these families were estimated using parsimony and distance-matrix approaches. All families appeared monophyletic. The Myricaceae appeared to derive first before the Betulaceae and the Casuarinaceae. In the Casuarinaceae, the genus Gymnostoma derived before the genera Casuarina and Allocasuarina, which were found closely related. The analysis of character-state changes in promiscuity along the consensus tree topology suggested a strong relationship between the evolutionary history of host plants and their promiscuity toward Frankia. Indeed, the actinorhizal taxa that diverged more recently in this group of plants were shown to be susceptible to a narrower spectrum of Frankia strains. The results also suggest that the ancestor of this group of plant was highly promiscuous, and that evolution has proceeded toward narrower promiscuity and greater specialization. These results imply that a tight relationship between the phytogenies of both symbiotic partners should not be expected, and that host promiscuity is likely to be a key determinant in the establishment of an effective symbiosis.     

The diversity of actinorhizal symbiosis

Filamentous aerobic soil actinobacteria of the genus Frankia can induce the formation of nitrogen-fixing nodules on the roots of a diverse group of plants from eight dicotyledonous families, collectively called actinorhizal plants. Within nodules, Frankia can fix nitrogen while being hosted inside plant cells. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. In this review, an overview of nodule induction mechanisms and nodule structure is presented including comparisons with the corresponding mechanisms in legume symbioses.     

尼泊尔马桑根瘤内生菌纯培养物的质粒检测

采用胶内裂解法快速检测了21株马桑根瘤内生菌纯培养物和4株弗兰克氏菌参考菌株的质粒,其中有5株马桑分离菌株和1株参考菌株含有质粒。除马桑菌株和参考菌株各有1株携带2个质粒外,其它菌株均只含有1个质粒。这些质粒的分子量约为13～20kb。根据所含质粒的大小和数目,将21株马桑分离菌株划分成4个质粒类群。实验还对菌丝体生长,细胞酶解和裂解等条件对质粒检测效果的影响进行了探讨。     

PCR-restriction fragment length polymorphism identification and host range of single-spore isolates of the flexible Frankia sp. strain UFI 132715.

Twelve single-spore isolates of the flexible Elaeagnus-Frankia strain UFI 132715 fulfilled the third and the fourth of Koch's postulates on both Alnus and Elaeagnus axenic plants. Seminested nifD-nifK PCR-restriction fragment length polymorphisms provided evidence for the genetic uniformity of the single-spore frankiae with the mother strain and its plant reisolates and allowed their molecular identification directly inside Alnus and Elaeagnus nodules. The clonal nature of these single-spore-purified frankiae should allow safe mutagenesis programs, while their flexible phenotype makes them a powerful tool for understanding the molecular interactions between Frankia strains and actinorhizal plants and for identifying Frankia nodulation genes.