Are Northwestern Patagonian “mallín” wetland meadows reservoirs of <Emphasis Type="BoldItalic">Ochetophila trinervis</Emphasis> infective <Emphasis Type="BoldItalic">Frankia?</Emphasis>

“Mallín” (plural mallines) is a particular kind of wetland occurring in Patagonian steppe and forests. In Northwest Patagonia, mallines are humid meadows with high net primary production. It was previously found that a mallín soil in the steppe devoid of actinorhizal plants had a higher Frankia nodulation capacity in Ochetophila trinervis (sin. Discaria trinervis) than other soils in the region. Under the hypothesis that mallín wetland meadows are reservoir of infective Frankia, we studied the Frankia nodulation capacity in O. trinervis of 12 mallín and their neighbouring steppe soils, by using plant bioassays. A qualitative plant bioassay showed that infective Frankia was present in most soils. The number of nodules per plant in seedlings inoculated with mallín soils was negatively correlated with soil water content while the opposite was true for plants inoculated with soils from neighbouring steppe. A quantitative bioassay was performed with eight representative soils, selected according to the number of nodules per plant produced in the qualitative assay and to the presence or not of different actinorhizal plants at the sites. Frankia nodulation units per cm³ of soil (NU) in mallín soils were higher than those in steppe. Water and organic matter content of soils were correlated with the higher nodulation capacity of mallines, which may account for the saprotrophic growth of Frankia in soils. The symbiosis was effective in plants inoculated with all soil samples. These results suggest that Northwestern Patagonian mallín wetland meadows are reservoirs of infective and effective Frankia propagules in O. trinervis.     

Effects of calcium in the nitrogen-fixing symbiosis between actinorhizal <Emphasis Type="Italic">Discaria trinervis</Emphasis> (Rhamnaceae) and <Emphasis Type="Italic">Frankia</Emphasis>

We have analysed the growth and symbiotic performance of actinorhizal Discaria trinervis at various Ca supply regimes. We aimed at discriminating between specific, if any, effects on nodulation and general growth stimulation by Ca. The hypothesis that a high Ca supply would interfere with nodulation by Frankia was also tested. Results showed that plant growth increased with Ca supply. Nodulation was stimulated by moderate levels of Ca, but inhibited by Ca higher than 0.77 mM. Growth of nodules was less affected by Ca than shoot and root growth. Ca concentration of symbiotic plants increased with Ca supply, but nitrogen concentration was independent of Ca at concentrations which did not impair plant growth. All together, these results show that Ca has a positive effect on the establishment and functioning of the symbiosis between Discaria trinervis and Frankia. However, the positive influence of Ca was more likely due to a promotion of plant growth rather than a direct effect on nodule growth and nitrogen fixation itself. At high levels of Ca supply nodulation was impaired. Given the intercellular infection pathway in Discaria trinervis, we suggest that the increment of Ca availability would strengthen its root cell walls, thus decreasing Frankia penetration of the root.     

Effects of Zn<Superscript>2+</Superscript> on nodulation and growth of a South American actinorhizal plant, <Emphasis Type="Italic">Discaria americana</Emphasis> (Rhamnaceae)

Discaria americana is a xerophytic shrub which lives in symbiosis with an actinomycete of the genus Frankia. The objective of this paper was to investigate the effects of high soil Zn²⁺ concentrations on growth and nodulation on the association Discaria americana–Frankia with the aim of determining if this association is suitable for improving contaminated soils. Two experiments were performed in 1 dm³ pots containing soil and different Zn additions, from 0 to 2,000 mg Zn²⁺ kg⁻¹ dry soil, with or without N fertilization. Zn additions strongly delayed shoot and root growth, but once growth was initiated, the biomass production of the plants supplied with moderate Zn amounts did not differ from the control plants. Zn reduced the final nodule number, but not the total nodule biomass. At the end of the experiment only the highest Zn treatments showed a lower nodule weight than the control plants, while N addition completely inhibited nodulation. It is concluded than Zn reduces the number of Frankia infections, but once the actinomycete is inside the roots, nodules can continue growing according to plant demand for N, compensating the reduced nodule number with more biomass. On the other hand, there is a toxic effect of Zn itself on plants when present in very high concentrations.     

Devising an <Emphasis Type="Italic">in vitro</Emphasis> nodulation system for two actinorhizal plants: <Emphasis Type="Italic">Allocasuarina verticillata</Emphasis> (Lam.) L. Johnson and <Emphasis Type="Italic">Casuarina glauca</Emphasis> sieb. ex spreng (Casuarinaceae)

Summary The purpose of this study was to establish an efficient in vitro nodulation device for producing actinorhizal root nodules on Allocasuarina verticillata and Casuarina glauca. Seeds from the two species were germinated aseptically and seedlings with at least two photosynthetic branchlets and a 3–5 cm long root system were transferred into Petri dishes containing a biphasic (solid/liquid) medium. To assess the nodulation capacity, four different culture media were tested. As soon as the root system developed and spread adequately on the surface of the medium, plants were deprived of nitrogen for at least 1 wk and inoculated with the Frankia strain. The time course nodulation for A. verticillata showed that the basal Hoagland medium supplemented with CaCO₃ and KNO₃ was most efficient, with 83% of plantlets forming nodules, while the medium supplemented with CaCO₃ reached 100% nodulation for C. glauca. This procedure can provide a valuable tool for the study of early events of actinorhizal nodulation and spatio-temporal expression of symbiotic genes in transgenic Casuarinaceae.     

The capacity of Jamaican mine spoils, agricultural and forest soils to nodulate Myrica cerifera, Leucaena leucocephala and Casuarina cunninghamiana

Soils from seven sites on the island of Jamaica were assayed for the symbiotic diazotrophs Frankia and Rhizobium using serial dilutions. Most probable number and least squares regression methods were used to estimate each soil's capacity to nodulate native Myrica cerifera, exotic Leucaena leucocephala and exotic Casuarina cunninghamiana. The sample sites included a montane forest, a slash-and-burn agricultural site, reclaimed bauxite mining areas, abandoned sugar cane fields, and a garden plot. None of the host plants used in the bioassay were present on the sites sampled except for scattered L. leucocephala on one site. Frankia capable of nodulating M. cerifera, which is native to Jamaican highlands, occurred at all sites sampled. No C. cunninghamiana-infective Frankia was detected in soils sampled. Only soils from one site on the tropical coastal plain harbored rhizobia able to nodulate L. leucocephala (37 nodulation units cm^?3 of soil). A subset of nodulated M. cerifera and L. leucocephala reduced acetylene to ethylene indicating nitrogenase activity. The slash-and-burn agricultural site, which was situated at an elevation of 200 m and possessed both high natural fertility and high soil moisture-supplying capacity, had significantly greater Myrica infectious capacity (1 000 nodulation units cm^?3 of soil) than the other sites (7?207 nodulation units cm^?3 of soil). A planned, paired comparison revealed that a recently cultivated sugar cane field and a recently reclaimed bauxite mining site together had significantly less Myrica-infective Frankia (4 nodulation units cm^?3 of soil) than a corresponding pair of sites consisting of a sugar cane field abandoned for 25 years and a bauxite mining site reclaimed 20 years before sampling (118 nodulation units cm^?3 of soil). Results indicate that Myrica-infective Frankia is widespread in Jamaica, that the number of Myrica-infective Frankia units vary from site to site in accordance with soil type and soil history, that Jamaican sites sampled lack soil Frankia populations capable of nodulating a casuarina host, that rhizobial symbionts capable of nodulating L. leucocephala may be geographically restricted to lowlands in Jamaica, and that the occurrence of Frankia in these soils is independent of host plant presence.     

Nodulation potential of soils from red alder stands covering a wide age range

Red alder (Alnus rubra Bong.) stands in the Pacific Northwest are the common first stage in succession following disturbance. These stands are highly productive and contribute a large amount of N to the soils as a result of their N₂-fixing symbiosis with Frankia. As these alder stands age, the soils not only increase in total N, but concentrations of NO^– ₃ increase and pH decreases as a result of nitrification. The objective of this study was to determine how the nodulation capacity of Frankia varies as red alder stands age and if differences in nodulation capacity are related to changes in soil properties. Nodulation capacity was determined by a red alder seedling bioassay for soils from red alder stands in the Oregon coast range covering a wide range of ages. Six chronosequences were sampled, each containing a young, an intermediate, and an older alder stand. Soil total N, total C, NO^– ₃, NH⁺ ₄, and pH were measured on the same soil samples. These factors as well as alder stand characteristics were compared with nodulation capacity in an attempt to identify soil characteristics typical in developing alder stands that most strongly affect nodulation capacity. Soil pH and NO^– ₃ concentration were highly correlated with nodulation capacity and with each other. Cluster analysis of the sites using these two variables identified two groups with distinctly different nodulation capacities. The cluster with the higher nodulation capacity was lower in NO^– ₃ and higher in pH than the other cluster, which included the majority of sites. There was substantial overlap in the age ranges for the two clusters and there was no significant correlation between age and nodulation capacity. Thus nodulation capacity appears to be most closely related to soil properties than to stand age.     

Feedback regulation of nodule formation in Hippophaë rhamnoides

Seedlings of Hippophaë rhamnoides possessing two equally infectible root systems (split roots) were used in conjunction with specific Frankia strains to investigate plant control over nodulation. When a wild-type Frankia strain was inoculated onto both root systems simultaneously or 1, 2, 4, or 8 weeks apart, an inhibitory response occurred which retarded nodulation on the root exposed to the delayed inoculum. Similar suppressive responses were also observed when two different wild-type Frankia inocula were applied onto opposite sides of a split-root system at different times. The depressed response shown by the delayed inoculum was more pronounced as the delay period was increased. The roots exposed to the delayed inoculum displayed a complete lack of nodulation when the delay was 4 or 8 weeks. The nodulation response on the root inoculated first depended on subsequent inoculation of the second root system of the plant, so that maximum nodulation of the first root was observed when the second root was unnodulated. These results provide evidence that sea buckthorn has an active, systemic mechanism for feedback control of nodulation that suppresses further nodule formation and prevents excessive nodulation. The significance of these results to the understanding of nodule ontogeny is discussed.     

Both the arbuscular mycorrhizal fungus Gigaspora rosea and Frankia increase root system branching and reduce root hair frequency in Alnus glutinosa

Alnus glutinosa is an important pioneer species that forms effective symbioses with Frankia and ecto and arbuscular mycorrhizal fungi (AMF). There is evidence that Frankia and AMF interact and the focus of this study was to investigate how interactions affected root system and root hair development. A. glutinosa seedlings were grown in pots in soil pre-inoculated with the AMF Gigaspora rosea. Seedlings were inoculated with Frankia either immediately on transfer to AMF-inoculated pots (day 0) on day 15 or on day 30 following AMF inoculation so the effect of timing of inoculation on interactions could be determined. Seedlings were harvested in batches at intervals of 10, 15, 20, 25 and 30 days after the commencement of each treatment. Both G. rosea and Frankia increased root branching and effects were greater when both were present. By contrast, both G. rosea and Frankia decreased root hair numbers markedly. Effects on root hair development were not a consequence of phosphorous, as P levels were not changed significantly in seedlings colonised by G. rosea or nodulated by Frankia. Effects are not due to differences in root system size but conceivably could offset some of the carbon costs incurred by the symbioses.     

Effect of <Emphasis Type="Italic">Pinus ponderosa</Emphasis> afforestation on soilborne <Emphasis Type="Italic">Frankia</Emphasis> and saprophytic Actinobacteria in Northwest Patagonia,Argentina

Large areas in the extra-Andean region in the forest - steppe ecotone in “Northwestern Argentinean Patagonia” have been replaced by plantations of the exotic conifer Pinus ponderosa which modify soils physical and chemical factors and alter the biodiversity. Considering that in the region occur saprophytic soilborne actinobacteria that play important role as the fixation of atmospheric nitrogen (N₂) in symbiosis with native plant species and the production of bioactive molecules in plants rhizosphere, we aimed to study the effect of the plantation on the abundance of the N₂ fixer Frankia and on the genus diversity of cultivable rhizospheric actinobacteria. The study was performed with soils of six paired sites with pine plantations and natural neighbor areas (including steppes or shrublands). Abundance of infective Frankia was estimated by evaluating the nodulation capacity of soils, through a plant bioassay using Ochetophila trinervis as trap plant. Isolation trials for saprophytic actinobacteria were performed by applying chemotactic and successive soils dilutions methods. We concluded that P. ponderosa afforestation affect soil actinobacteria. This was mainly evidenced by a decrease in the Frankia nodulation capacity in O. trinervis, which was related to plantation age, to lower soil carbon and nitrogen content, higher available phosphorus, and to a slight decrease in soils pH. Pine plantation influence on the cultivable saprophytic actinobacteria was less clear. The study highlights the importance of soils as source of Frankia and rhizospheric actinobacteria in relation to disturbance caused by pine plantation in natural environments with native actinorhizal plant species.     

Effect of Inoculation and Leaf Litter Amendment on Establishment of Nodule-Forming Frankia Populations in Soil

High-N₂-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 [¹⁵N]NO₃ 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 N₂ 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 N₂ 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 N₂ fixation rates by ¹⁵N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N₂-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 N₂-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.     

Survival ofFrankia strains introduced into soil

Factors affecting the establishment of Alnus/Frankia symbioses were studied partly by following the survival ofFrankia strains exposed to different soil conditions, and partly by investigating the effect of pH on nodulation. TwoFrankia strains were used, both of the Sp⁻ type (sporangia not formed in nodules). One of the strains sporulated heavily, while the other formed mainly hyphae. The strains originated fromAlnus incana root nodules growing in soils of pH 3.5 and 5.0. The optimum pH for their growth in pure culture was found to be 6.7 and 6.2, respectively. The strains were introduced into twoFrankia-free soils, peat and fine sand. Their survival, measured as the persistance of nodulation capacity using the plant infection technique, was followed for 14 months. The survival curves of the strains were similar despite the morphological differences between the strains in pure culture. The nodulation capacities declined over time both at 14 and 22°C. Survival was better in soils limed to a pH above 6 than in soils at their original pH (peat 2.9, fine sand 4.2). The effect of pH on nodule formation in Alnus seedlings by theFrankia strains was studied in liquid culture. The number of nodules increased linearly within the pH range studied (3.5–5.8). No nodules were formed at pH 3.5.     

基于高通量测序分析西藏沙棘根瘤内生菌的多样性

根瘤是微生物侵染植物根部并与之形成的共生结构,这些微生物都可被称为植物内生菌。豆科植物根瘤中的内生菌常常又被称为根瘤菌,而侵染非豆科植物形成根瘤的主要是放线菌弗兰克氏菌,这些非豆科植物又被称为放线菌结瘤植物。西藏沙棘是一种典型的放线菌结瘤植物,由于其分布生境的特殊性,对其根瘤内生菌的研究具有重要的生态意义。对于西藏沙棘根瘤内生菌的研究,培养方法因难以模拟自然条件而不易获得纯培养,高通量测序技术对其多样性的研究提供了便利。因此,本研究以生长在甘肃省天祝县金强河河滩地的西藏沙棘根瘤为材料,采用16S rRNA基因扩增子高通量测序方法,结合OTU分析,对西藏沙棘根瘤内生菌的多样性进行探讨。实验结果表明,西藏沙棘根瘤内生菌具有丰富的多样性,根瘤内的优势属为共生固氮的弗兰克氏菌属（Frankia）,其相对丰度为47.63%,共检测到7个弗兰克氏菌属的OTUs;根瘤内除弗兰克氏菌外,还存在大量的非弗兰克氏菌,共检测到1523个OTUs,隶属于22个门、33个纲、69个目、113个科和202个属,相对丰度排名前9的属中有25个非弗兰克氏菌属的OTUs。该研究也表明,西藏沙棘根瘤内生菌具有丰富的多样性,西藏沙棘根瘤中不仅存在着可共生固氮的弗兰克氏菌,并且还分布着非弗兰克氏菌;在同一根瘤样品中,弗兰克氏菌属还具有不同的物种。本研究不仅拓展了西藏沙棘根瘤内生菌多样性的研究方法,还为同一寄主植物中弗兰克氏菌多样性的研究提供了分析思路。     

Frankia inoculation,soil biota,and host tissue amendment influence Casuarina nodulation capacity of a tropical soil

The effects of soil biota, Frankia inoculation and tissue amendment on nodulation capacity of a soil was investigated in a factorial study using bulked soil from beneath a Casuarina cunninghamiana tree and bioassays with C. cunninghamiana seedlings as capture plants. Nodulation capacities were determined from soils incubated in sterile jars at 21 °C for 1, 7, and 28 days, after receiving all combinations of the following treatments: ± steam pasteurization, ± inoculation with Frankia isolate CjI82001, and ± amendment with different concentrations of Casuarina cladode extracts. Soil respiration within sealed containers was determined periodically during the incubation period as a measure of overall microbial activity. Soil respiration, and thus overall microbial activity, was positively correlated with increasing concentrations of Casuarina cladode extracts. The nodulation capacity of soils inoculated with Frankia strain Cj82001 decreased over time, while those of unpasteurized soils without inoculation either increased or remained unaffected. The mean nodulation capacity of unpasteurized soil inoculated with Frankia CjI82001 was two to three times greater than the sum of values for unpasteurized and inoculated pasteurized soils. Our results suggest a positive synergism between soil biota as a whole and Frankia inoculum with respect to host infection.     

An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

An ineffective strain type of Frankia of unknown strain composition, coded AgI-WD1 was discovered in the soil of wet dune slacks where A. glutinosa was the dominant tree species. Strain type AgI-WD1 was recognized by the development of slow growing root nodules on A. glutinosa testplants inoculated with soil suspensions. Microscopical examination of these nodules showed extremely reduced development of vesicles, normal development of intracellular clusters of hyphae and absence of sporangia. The stability of characteristics of this strain type such as the expression of root nodule symbiosis and ineffectivity of symbiontic N-fixation was demonstrated through ‘subculture’ of ineffective root nodules in successive hydrocultures of A. glutinosa. The nodulation process also differed from normal effective root nodules by the occurrence of resistance to strain type AgI-WD1 among part of the half-siblings of A. glutinosa used in the nodulation tests. Strain type AgI-WD1 was detected in the soil of different dune slacks which are inundated for a large part of the year and in a nearby peatbog covered with alder. The contribution of this strain type to soil populations of Frankia was demonstrated by nodulation potentials that were up to 500 times higher than that of the concurrent effective strain type AgSp^-. The distribution of strain type AgI-WD1 appeared to be restricted to sites with water-logged soil conditions. Nodulation experiments pointed to potentials for competitive interactions between effective and ineffective strain thpes, especially to a density dependent reduction of nodule type AgI-WD1 by strain type AgSp^-. The impact of competitive interactions is also affected by host trees that are resistant to AgI-WD1. The occurrence of resistance in the study areas was suggested by resistance among seedlings of a local seedbatch (±70% of the half-siblings) and by the absence of ineffective root nodules at site VD7-1, despite a high nodulation potential of the soil population of strain type AgI-WD1.     

Ineffective Frankia and host resistance in natural populations of Alnus glutinosa (L.) gaertn

Alnus glutinosa (black alder) populations are known to exhibit a variable degree of incompatibility to root nodule formation by ineffective Frankia. The relationship between the occurrence of ineffective Frankia in wet stands of black alder and the degree of resistance to nodulation by ineffective Frankia of seed-lots and clones of alder trees from these particular locations was studied through soil inoculation experiments. The average percentage of resistant plants (R-frequency) among the seed-lots from locations with an ineffective Frankia soil population was equal to, or higher than, the R-frequencies of locations without ineffective Frankia. The mean R-frequency was highest for the seed-lots from the location from which the soil inoculant was taken. These results strongly suggest that ineffective Frankia are not strictly dependent on susceptible A. glutinosa for the maintenance of their population size. The fungus Penicillium nodositatum also nodulated A. glutinosa seedlings. Whereas a negative interaction with the ineffective Frankia nodulation was found, this did not have a significant effect on the R-frequencies of the seed-lots that were tested, suggesting that the ineffective Frankia nodulation adversely affected the myco-nodulation, and not vice versa.     

The Actinorhizal Symbiosis

Abstract The term ``actinorhiza' refers both to the filamentous bacteria Frankia, an actinomycete, and to the root location of nitrogen-fixing nodules. Actinorhizal plants are classified into four subclasses, eight families, and 25 genera comprising more than 220 species. Although ontogenically related to lateral roots, actinorhizal nodules are characterized by differentially expressed genes, supporting the idea of the uniqueness of this new organ. Two pathways for root infection have been described for compatible Frankia interactions: root hair infection or intercellular penetration. Molecular phylogeny groupings of host plants correlate with morphologic and anatomic features of actinorhizal nodules. Four clades of actinorhizal plants have been defined, whereas Frankia bacteria are classified into three major phylogenetic groups. Although the phylogenies of the symbionts are not fully congruent, a close relationship exists between plant and bacterial groups. A model for actinorhizal specificity is proposed that includes different levels or degrees of specificity of host-symbiont interactions, from fully compatible to incompatible. Intermediate, compatible, but delayed or limited interactions are also discussed. Actinorhizal plants undergo feedback regulation of symbiosis involving at least two different and consecutive signals that lead to a mechanism controlling root nodulation. These signals mediate the opening or closing of the window of susceptibility for infection and inhibit infection and nodule development in the growing root, independently of infection mechanism. The requirement for at least two molecular recognition steps in the development of actinorhizal symbioses is discussed.     

Is the Legume Nodule a Modified Root or Stem or an Organ sui generis?

The legume nodule, which houses nitrogen-fixing rhizobia, is a unique plant organ. Its homology with lateral roots has been inferred by a comparison with other nitrogen-fixing nodules, especially those formed on actinorhizal plants in response to Frankia inoculation or on Parasponia roots following inoculation with Bradyrhizobium species. These nodules are clearly modified lateral roots in terms of their structure and development. However, legume nodules differ from lateral roots and these other nodules in their developmental origin, anatomy, and patterns of gene expression, and, consequently, several other evolutionary derivations, including from stems, wound or defense responses, or the more ancient vesicular-arbuscular mycorrhizal symbiosis, have been postulated for the legume nodule. In this review, we first present a broad view of the legume family showing the diversity of nodulation occurrence and types in the different subfamilies and particularly within the subfamily Papilionoideae. We then define the typological and molecular criteria used to discriminate the basic organs — root, stem, leaf— of the plant. Finally, we discuss the possible origins of the legume nodule in terms of these typological and molecular bases.     

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 N₂-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.     

Bacteria-plant interactions in symbiotic nitrogen fixation

In the inter- and intracellular N₂-fixing symbioses between plants and micro-symbionts, the development of an endophytic form of the micro-symbiont is essential. This development includes a series of steps consisting of plant-bacteria interactions. Considerable progress in the elucidation of these steps has been made by applications of the methods of molecular genetics. Several genes with a role during infection and nodulation have been indicated in Rhizobium and Bradyrhizobium like the common nod genes A, B, C, I and J, and the host-specific genes nod E, F and H. The nod D gene is the only constitutive gene, and its product is essential for activity of all other nod genes, provided some flavonoids from the root exudate are present as well. Mutants in these genes show phenotypic effects, in which the products of the genes must be involved. Far more difficult is the biochemical and physiological study of these products and their direct effects. The difficulties involved in such biochemical-physiological studies is illustrated by a short discussion of the controversies around the possible role of plant lectins. While in Rhizobium the nod genes are present on a large sym-plasmid, other essential genes must be present on the bacterial chromosome and on other plasmids. Induction of plant genes is evident from the formation of nodule-specific proteins, the nodulins. Though many different plant and bacterial genes are involved in the series of steps in the development of an effective root nodule, there are indications that regulation is affected by a smaller number of essential regulatory genes. This is illustrated by the effect of the regulatory nod D gene during infection and nodulation, and of ntrA and nifA genes for the formation and activation of the nitrogen-fixing systems. Moreover, every step, once initiated, may lead to cascade effects on subsequent reactions. Finally, some further consequences of the endophytic way of life are discussed, which affect either the metabolic and transport activities of the endophytes or their viability. This is illustrated by the possible role of membrane integrity as evident during the isolation of Frankia from its endophytic form.     

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.