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.     

Transformed hairy roots of Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.     

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.     

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

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.     

Local Adaptation of Frankia to Different Discaria (Rhamnaceae) Host Species Growing in Patagonia

Frankia BCU110601 (Da) and Frankia BCU110345 (Dc) were isolated from root nodules of Discaria articulata and Discaria chacaye, respectively; Frankia BCU110501 (Dt) was previously isolated from Discaria trinervis. The strains were identical at the 16S sequence and after analysis of RFLP of 16S and 23S rDNA intergenic region. Diversity was revealed at the molecular level after fingerprint analysis by BOX–polymerase chain reaction. The strains were infective and effective on the original host plants. A cross-inoculation assay intra Discaria genus, including D. trinervis, D. articulata, and D. chacaye, with each of these isolated Frankia strains caused effective symbioses with a similar dry weight in each plant species regardless of the inoculated strain. Nevertheless, a differential degree of recognition was revealed: Homologous symbiotic pairs in the case of D. chacaye–Frankia BCU110345 (Dc), D. articulata–Frankia BCU110601 (Da), and D. trinervis–Frankia BCU110501 (Dt) had faster nodulation rates than heterologous pairs. The differences in nodulation rate would suggest the existence of a subspecific level of recognition within a certain cross-inoculation group, pointing to subspecific adaptation occurring in this actinorhizal symbiosis.     

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.     

Nitrogen fixation and biomass production in symbioses between Alnus incana and Frankia strains with different hydrogen metabolism

Three different strains of Frankia , the pure cultures AvcI1 and CpI1 and a local strain (crushed nodule inoculum), were compared in symbiosis with one clone of Alnus incana (L.) Moench. Hydrogen metabolism, nitrogenase (EC 1.7.99.2) activity and relative efficiency of nitrogenase were studied as well as growth and nitrogen content of the plants. The local Frankia strain showed no measurable hydrogen uptake but high H₂-evolution. No H₂-evolution was detected in Frankia AvcI1 because of its hydrogenase activity. CpI1 also had hydrogenase, although only a very small H₂-evolution was detected at the end of the growth period. Hydrogenase activity was detected both in pure cultures and nodule homogenates of CpI1 and AvcI1. Growth, biomass production and nitrogen content were highest in alders inoculated with Frankia AvcI1 while the lowest values were found for alders living in symbiosis with the local Frankia strain. The presence of hydrogenase in Frankia seemed to be benefical for growth and biomass production in the alders. However, the strains also differed with respect to spore formation. The local strain, but not AvcI1 and CpI1, formed spores in the root nodules.     

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.     

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.     

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.     

The abilities of three Frankia isolates to nodulate and fix nitrogen with four species of Casuarina

Three isolates of Frankia from nodules of Casuarina sens, strict. (JCT287. JCT295 and 20607) were compared in their abilities to nodulate and fix N, when associated with four species of Casuarina ( C. cunninghamiana Miq., C. equisetifolia Forst., C obesa Miq. and C. glauca Sieb. ex Spreng) growing in a N-deficient soil.
All three Frankia isolates nodulated each of the four species of Casuarina . At 27 weeks after inoculation, growth (dry weight) of inoculated plants was 3.6 to 5.0 times greater than that of uninoculated plants. There were no significant differences in plant dry weight, the N concentration of shoots or roots, or the amount of N, fixed per plant among the Frankia isolates for each of the species of Casuarina studied. The infectivity and effectiveness in N, fixation of Frankia strain JCT287 with C. cunninghamiana was similar when two different defined media were used for culture of the inoculum.     

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.     

Hemoglobin in five genetically diverse Frankia strains

Five strains of Frankia were selected to represent a wide range of genetic diversity and examined for presence of hemoglobin. All five strains produced hemoglobin when grown on media without (-N) or with (+N) combined nitrogen. This indicates that hemoglobin is common in Frankia and is not directly associated with nitrogen fixation. Frankia strain EAN1(pec) was examined in more detail. It showed greater hemoglobin concentration when grown at 2% O2 than at 20% O2 in the -N treatment but no effect of oxygen on hemoglobin concentration in the +N treatment. At both oxygen levels, it produced substantially more biomass in +N than in -N culture. It also produced significantly more biomass when the medium contained 0.2% CO2 than in the absence of CO2. The molecular mass of the hemoglobin as determined by size exclusion chromatography was 13.4 +/- 0.2 kDa (mean +/- SE, n = 3) and is consistent with that of a truncated hemoglobin. The hemoglobin had absorption spectra that were typical of a hemoglobin. The oxygen dissociation rate constants for the hemoglobin were 131.2 +/- 5.8 s(-1) for -N culture and 166 +/- 8.2 s(-1) for +N culture. These rapid rates are consistent with a function in facilitated diffusion of oxygen.     

沙棘属植物弗兰克氏菌研究进展

弗兰克氏菌(Frankia spp.)能够与沙棘等非豆科植物形成根瘤进行共生固氮,其固氮效率远远高于豆科植物根瘤菌,与沙棘共生的弗兰克氏菌还能够促进沙棘对旱寒等各种不同生境的适应性,是自然界一类具有开发潜力的放线菌资源。为了更好地开发利用弗兰克氏菌资源,推进弗兰克氏菌分类鉴定工作,加强弗兰克氏菌与寄主植物共生结瘤固氮的机制研究,促使弗兰克氏菌在农业生产中得到尽快应用,本文简要介绍沙棘属(Hippophae L.)物种多样性、结瘤状况与分布特点、沙棘根瘤形态结构与功能、弗兰克氏菌物种多样性与分布特征,讨论弗兰克氏菌的结瘤机制、生理生态效应与作用机制以及影响沙棘属植物与弗兰克氏菌共生的主要因子,以期为进一步开展沙棘属植物弗兰克氏菌的系统研究提供有价值的参考。     

A reconsideration of terminology in Frankia research: A need for congruence

Progress in understanding the Frankia -actinorhizal symbiosis has paralleled that of the Rhizobium -legume symbiosis. Previously, these two nitrogen-fixing symbioses have been considered as distinct and unrelated entities. However, it seems likely that many of the developmental stages, and perhaps even the molecular signals, are shared between these two apparently independent associations. For this reason, we propose to emphasize the similarities between the two symbioses by calling for a uniformity of terminology to describe nodule development.     

Water-retentive polymers increase nodulation of actinorhizal plants inoculated with Frankia

Actinorhizal plants form a nodular, nitrogen-fixing root symbiosis with the actinomycete Frankia and are economically and ecologically important due to their ability to improve the nitrogen fertility of disturbed and infertile substrates. In this study, water-retentive polymer inoculum carriers were applied as a root dip. This treatment significantly increased nodulation and in some cases early growth of Alnus glutinosa (L.) Gaertn. and Casuarina equisetifolia var. equisetifolia Forst. & Forst. in a controlled environment and also of A. glutinosa under field conditions. Nodule number and nodule dry weight per plant were at least two to three times greater after 56 to 140 days for plants inoculated with Frankia carried in a water-retentive polymer base compared with plants inoculated with Frankia in water. Nodules on the roots of the plants that were inoculated with Frankia in a polymer slurry were distributed throughout the entire root system, rather than concentrated near the root collar. When amended with water-retentive polymers, actinorhizal plants inoculated with 5- to 10-fold lower titers of Frankia exhibited early growth and nodule numbers equal to or greater than those plants inoculated with standard titers without polymers. The water-retentive, superabsorbent polymers clearly increased the nodulation of two actinorhizal plant species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of symbiosis with Frankia and arbuscular mycorrhizal fungus on the natural abundance of 15N in four species of Casuarina

The effect of interactions between Casuarina species, Frankia strains and AMF on nitrogen isotope fractionation within the plant were determined under conditions where changes in source nitrogen were minimized by growing plants in mineral nitrogen-deficient conditions and without added organic N. Casuarina cunninghamiana, C. equisetifolia, C. glauca, and C. junghuniana were inoculated singly with three Frankia strains or were dual inoculated with Frankia and Glomus fasciculatum. The %N and delta 15N of separated parts of plants inoculated with the three Frankia strains or with Frankia + Glomus were not significantly different within Casuarina species. However, the slow-growing C. junghuniana differed in several variables from the other three species. There was a highly significant, linear relationship between the natural logarithms of cladode N content and delta 15N of plants of the four Casuarina species when inoculated with Frankia or with Frankia + Glomus, showing that nitrogen supply and the correlated variable, plant growth rate, were major determinants of delta 15N. Provision of small quantities of (NH4)2SO4 or KNO3 increased several-fold the growth of three of the Casuarina species when inoculated with Frankia alone or with Frankia + Glomus. Within species, mycorrhizal and non-mycorrhizal plants receiving supplementary soluble phosphate were of similar dry weights at harvest. delta 15N values for cladodes of C. cunninghamiana, C. equisetifolia and C. glauca were similar, but values for the poor growing C. junghuniana were more variable and, with the exception of plants receiving KNO3, were lower than those of the other three species. Reduced growth due to suboptimal availability of N or P had a major influence on delta 15N and, in these conditions where plants could not access significant amounts of organic N, outweighed any effects on cladode delta 15N of colonization by Glomus. delta 15N values of nodules were higher than other parts of Frankia or Frankia + Glomus inoculated Casuarinas, conceivably due to retention in nodules of fixed N, with delta 15N close to zero.     

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.