The nodular endophytes of Coriaria spp. form a distinct lineage within the genus Frankia

Repeated attempts at isolating the Frankia endophyte of Coriaria spp. have not yielded infective microbial cultures that could fulfil Koch's postulates. In order to circumvent the critical isolation step, nodule endophytes of Coriaria were characterized directly by means of specific amplification of nodule DNA (PCR) followed by sequencing of part of the 16S rDNA gene. Three closely related sequences were obtained from nodules originating from France, Mexico and New Zealand, containing unique sequences different from all other Frankia strains characterized so far. The sequences obtained were closest (with 5 or 6 substitutions) to those of Frankia alni and those of Casuarina-infective Frankia strains, respectively. Two nucleotides unique to the Coriaria endophyte sequences were used to define specific primers, resulting in a hybridization test that could discriminate between Frankia DNAs originating from Coriaria nodules and those recovered from all cultured Frankia strains tested. The endophytes of Coriaria thus appear to form a distinct Frankia lineage.     

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.     

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.     

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 .     

Regulation of nodulation in the absence of N2 is different in actinorhizal plants with different infection pathways

Root nodulation in actinorhizal plants, like Discaria trinervis and Alnus incana, is subject to feedback regulatory mechanisms that control infection by Frankia and nodule development. Nodule pattern in the root system is controlled by an autoregulatory process that is induced soon after inoculation with Frankia. The final number of nodules, as well as nodule biomass in relation to plant biomass, are both modulated by a second mechanism which seems to be related to the N status of the plant. Mature nodules are, in part, involved in the latter process, since nodule excision from the root system releases the inhibition of infection and nodule development. To study the effect of N(2) fixation in this process, nodulated D. trinervis and A. incana plants were incubated under a N(2)-free atmosphere. Discaria trinervis is an intercellularly infected species while A. incana is infected intracellularly, via root hairs. Both symbioses responded with an increment in nodule biomass, but with different strategies. Discaria trinervis increased the biomass of existing nodules without significant development of new nodules, while in A. incana nodule biomass increased due to the development of nodules from new infections, but also from the release of arrested infections. It appears that in D. trinervis nodules there is an additional source for inhibition of new infections and nodule development that is independent of N(2) fixation and nitrogen assimilation. It is proposed here that the intercellular Frankia filaments commonly present in the D. trinervis nodule apex, is the origin for the autoregulatory signals that sustain the blockage of initiated nodule primordia and prevent new roots from infections. When turning to A. incana plants, it seems likely that this signal is related to the early autoregulation of nodulation in A. incana seedlings and is no longer present in mature nodules. Thus, actinorhizal symbioses belonging to relatively distant phylogenetic groups and displaying different infection pathways, show different feedback regulatory processes that control root nodulation by Frankia.     

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.     

The use of PCR-based typing methods to assess the diversity of Frankia nodule endophytes of the actinorhizal shrub Ceanothus

Our understanding of the actinorhizal symbiosis, in particular of the Frankia-Ceanothus association, has been hampered by the failure to isolate infective strains in pure culture. Recently, the polymerase chain reaction (PCR) has been utilized to amplify regions of the Frankia genome, allowing analysis of the microsymbiont genome without first isolating the microbe in pure culture. Root nodules were collected from six Ceanothus spp. common to the coastal regions of the Santa Monica Mountains of southern California. Individual lobes were surface-sterilized, total DNA was extracted and amplified using prokaryotic-specific primers. To assess the genetic diversity of Frankia endophytes in the population studied, the BOX primer was used to generate genomic fingerprints of prokaryotic nodule inhabitants using rep-PCR. Fingerprint patterns fell into twelve distinct groups indicating the occurrence of genetic diversity of Frankia in the nodules sampled. DNA extracts of individual lobes that gave distinct BOX-PCR fingerprints were also amplified by PCR using primers directed against conserved regions of the 16S ribosomal RNA gene. The nucleotide sequences of the PCR products were determined and aligned with the corresponding region from other taxa for phylogenetic analysis. The sequences from Ceanothus nodules share a common ancestor to that of the Elaeagnus –infective strains.     

Natural diversity of Frankia strains in actinorhizal root nodules from promiscuous hosts in the family Myricaceae.

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N(2)-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.     

Time course of nodule development in the Discaria trinervis (Rhamnaceae) –Frankia symbiosis

The time course of initiation and development of root nodules was investigated in the South American actinorhizal shrub Discaria trinervis (Rhamnaceae). A local strain of Frankia (BCU110501) which was isolated from D. trinervis nodules, was used as inoculum. Inoculated seedlings were periodically studied under the light microscope after clearing with aqueous NaClO. In parallel, semithin and ultrathin sections were analysed by light and electron microscopy. Infection by Frankia BCU110501 involved intercellular penetration among epidermal and cortical root cells. Nodule primordia were detected from 6 d after inoculation, while bacteria were progressing through intercellular spaces of the outer layers of cortical cells. Invasion of host cells by the symbiont occurred 7–9 d after inoculation, and hypertrophy of the primordium cells was associated with Frankia penetration. Root hairs were not deformed during the early events of nodule formation. From 13 to 16 d after inoculation, the proximal cellular zone of the primordia behaved differently from the other tissues after NaClO treatment and remained darkly pigmented. At the same time, differentiation of Frankia vesicles started to occur inside already infected cells. By 16 d after inoculation, spherical vesicles of BCU110501 were homogeneously distributed in the host cells. These vesicles were septate and surrounded by void space. Frankia spores or sporangia were not observed in the nodule tissue. This study has clarified the mode of Frankia penetration in D. trinervis , one of the Rhamnaceae which also includes Ceanothus . The events involved in infection, nodule induction, host-cell infection and vesicle differentiation have been characterized and identified as time-segregated developmental processes in the ontogeny of D. trinervis root 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.     

Laser-based micromanipulation for separation and identification of individual Frankia vesicles

In studies of symbiotic efficiency it is of great importance to identify and separate individual Frankia strains from a nodule. Therefore, a new laser-based micromanipulation technique has been developed in which individual vesicles from root nodules of two Frankia-Alnus symbioses have been successfully cut loose and separated from clusters of vesicles in sterile conditions under light microscopy using a laser scalpel and optical tweezers. Vesicles from the Alnus incana-Frankia AvCI1 symbiosis were successfully isolated and grown in culture using this technique. The DNA from both Frankia sources was amplified by polymerase chain reaction (PCR). The work shows that a combination of laser-based manipulation techniques and PCR can be used for the separation and study of individual vesicles. This novel laser-based micromanipulation technique opens up various new possibilities, for instance, to study whether several Frankia strains can grow simultaneously in the same root nodule.     

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.     

Isolation and characterization of Frankia from nodules of actinorhizal plants of Pakistan

Frankia strains have been isolated from actinorhizal nodules of Alnus (2 strains), Casuarina (5 strains), Coriaria (1 strain), Datisca (3 strains), Elaeagnus (1 strain) and Hippophae (1 strain). The isolates were characterized for their growth on various carbon and nitrogen sources, nitrogen-fining ability in culture and nodulation of seedlings of the original host plant.     

Isolation of Elaeagnus-compatible Frankia from soils collected in Tunisia

The occurrence and diversity of Frankia nodulating Elaeagnus angustifolia in Tunisia were evaluated in 30 soils from different regions by a Frankia-capturing assay. Despite the absence of actinorhizal plants in 24 of the 30 soils, nodules were captured from all the samples. Eight pure strains were isolated from single colonies grown in agar medium. On the basis of 16S rRNA and GlnII sequences, seven strains were clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic groups while one strain described a new lineage in the Frankia assemblage, indicating that Frankia strains genetically diverse from previously known Elaeagnus-infective strains are present in tunisian soils. Genomic fingerprinting determined by rep-PCR, and tDNA-PCR-SSCP, confirmed the wide genetic diversity of the strains.     

高黎贡山旱冬瓜Frankia的IGS PCR-RFLP分析

在云南省高黎贡山自然保护区海拔1310～2400m的范围内,采集30个旱冬瓜根瘤样品,直接从根瘤中提取Frankia DNA,对其,nifD-nifK基因间隔区(intergenic spacer,IGS)和16S-23S rDNA IGS进行PCR—RFLP分析．结果表明,nifD-nifK IGS的PCR产物长度差异很大,经HaeⅢ和Afa I双酶切后,得到15种酶切带型,检测到多种基因型的菌株同时与同一株宿主植物共生;16S-23S rDNA IGS的PCR产物长度相似,酶切后亦区分出15种酶切带型．通过对两个基因间隔区的PCR-RFLP联合分析,发现高黎贡山旱冬瓜Frankia存在20种基因型．     

Frankia genus-specific characterization by polymerase chain reaction.

The polymerase chain reaction (PCR) is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. In order to determine whether a given actinomycete isolated from an actinorhiza (nodule) belongs to the genus Frankia or is a contaminant, we have developed a test based on the PCR. Primers complementary to sequences of two DNA regions corresponding to the nif genes (nifH and nifD) and the rRNA genes (16S and 23S) were specifically chosen to differentially amplify DNAs from Frankia strains but not those from other microorganisms. A series of positive and negative controls were set up by using universal or selective primers resulting in a discriminant amplification, which could be detected after agarose gel electrophoresis. In the nif region, degenerate oligonucleotide primers were used to amplify a target common to all the nitrogen-fixing microorganisms tested, while another set of primers amplified a target with a high specificity for Frankia strains. In the rRNA gene region, universal and specific primers were characterized and tested with DNAs from a wide range of microorganisms. The efficiency of this rapid and sensitive PCR assay was tested with an isolate obtained from Alnus nepalensis nodules, confirming results obtained by nodulation tests.     

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.     

Frankia genus-specific characterization by polymerase chain reaction.

The polymerase chain reaction (PCR) is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. In order to determine whether a given actinomycete isolated from an actinorhiza (nodule) belongs to the genus Frankia or is a contaminant, we have developed a test based on the PCR. Primers complementary to sequences of two DNA regions corresponding to the nif genes (nifH and nifD) and the rRNA genes (16S and 23S) were specifically chosen to differentially amplify DNAs from Frankia strains but not those from other microorganisms. A series of positive and negative controls were set up by using universal or selective primers resulting in a discriminant amplification, which could be detected after agarose gel electrophoresis. In the nif region, degenerate oligonucleotide primers were used to amplify a target common to all the nitrogen-fixing microorganisms tested, while another set of primers amplified a target with a high specificity for Frankia strains. In the rRNA gene region, universal and specific primers were characterized and tested with DNAs from a wide range of microorganisms. The efficiency of this rapid and sensitive PCR assay was tested with an isolate obtained from Alnus nepalensis nodules, confirming results obtained by nodulation tests.     

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。     

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.