Effective Frankia Research Articles

Are Northwestern Patagonian “mallín” wetland meadows reservoirs of Ochetophila trinervis infective Frankia?

“Mallin” (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 mallin 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 mallin wetland meadows are reservoir of infective Frankia, we studied the Frankia nodulation capacity in O. trinervis of 12 mallin 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 mallin 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 mallin 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 mallin wetland meadows are reservoirs of infective and effective Frankia propagules in O. trinervis.

Isolation of endophytic Frankia from root nodules of Casuarina equisetifolia and infectivity of the isolate to host plants

Attempts were made to isolate endophytic Frankia from the root nodules formed on Casuarina equisetifolia grown in a mixture of vermiculite and field soil obtained from the Okayama University Campus. The isolation of Frankia was performed by macerating a nodule lobe directly in a test tube containing modified Qmod medium with casamino acids and sodium pyruvate as nitrogen and carbon sources. One colony, which showed hyphal and sporangial characteristics similar to those of Frankia, appeared in a test tube after 1-month incubation. The root nodules were successfully formed on the roots of the host plants by inoculation of the isolate which was multiplied using the original Qmod medium containing yeast extract, peptone and propionate. The acetylene reduction activity (ARA) obtained was 6.29 ± 1.57 µmol C2H4 h−1 (g F.W. nod.)−1. Accordingly, we identified the isolate as an effective Frankia strain for C. equisetifolia and designated it as Ceq1. Since this strain also formed root nodules on Alnus sieboldiana and Myrica rubra, it was considered to have a wider host range than that of Frankia strains which had been previously isolated from Casuarina nodules. However, the values of the nodule fresh weight and ARA in A. sieboldiana and M. rubra were much lower than those in C. equisetifolia and only half of the plants tested were nodulated in M. rubra. This strain did not form root nodules on Elaeagnus macrophylla. Frankia vesicles, which are the specialized structures where nitrogenase is located, were not observed in the free-living culture grown in Qmod medium containing abundant nitrogen and in the nodules newly formed by inoculation of the Ceq1 strain.

Frankia and nodulation of red alder and snowbrush grown on soils from Douglas-fir forests in the H.J. Andrews experimental forest of Oregon

This study examined colonization of Frankia on actinorhizal red alder ( Alnus rubra) and snowbrush ( Ceanothus velutinus) in soils from three stands located at the H.J. Andrews experimental forest in the western Cascade Range of Oregon: an 8-year-old clear-cut planted with Douglas-fir ( Pseudotsuga menziesii), a young 20-year-old Douglas-fir plantation with an understory of snowbrush, and an old-growth Douglas-fir forest. Ten soil samples were collected from within each of the three stands; in each of these soils, plants were grown in a mix of soil–vermiculite–perlite (2:1:1). Alder plants were grown for 6 months and snowbrush for a year. More alder than snowbrush survived and nodulated. Of the plants that survived, 89% of the red alder nodulated; only 25% of the snowbrush produced nodules. More red alder plants nodulated when grown in clear-cut soils (100%) than in other soils, and more snowbrush nodulated (51%) when grown in soils from the 20-year-old plantation. Red alder biomass and nodule weight were highest when plants were grown in clear-cut soils. Snowbrush biomass and nodule weight were highest on soils from the young stand. The biomass of snowbrush plants grown in clear-cut soils averaged higher in bottom slope soils than in soils from any other position within the clear-cut. Correlations between plant biomass, nodule weight, and acetylene reduction activity were positive and statistically significant for both species. Differences in Frankia with regard to red alder and snowbrush were apparent in these study sites. The limited nodulation in snowbrush might have been due to an insufficient population of an effective Frankia population. Establishing snowbrush on this study site will require developing techniques for inoculating snowbrush seedlings with the relevant Frankia and then outplanting the inoculated seedlings on the sites.

Isolation of infective and effective Frankia strains from root nodules of Alnus acuminata (Betulaceae).

Two Frankia strains were isolated from root nodules of Alnus acuminata collected in the Tucumano-oranense forest, Argentina. Monosporal cultures were obtained by plating a spore suspension of each strain and isolating a single colony. The strains (named AacI and AacIII) showed branched mycelia with polymorphic sporangia and NIR-vesicles. They differed in their ability to use carbon sources: the AacI strain grew well on pyruvate, while the AacIII strain grew on mineral medium supplemented with glucose or, alternatively, with sucrose. The two strains were sensitive to oleandomycin, erythromycin, kanamycin, penicillin G, streptomycin and chloramphenicol at 5 μg/ml. The AcIII strain exhibited a moderate resistance to rifampicin, ampicillin and vancomycin. The nitrogenase activity in vitro of the strains was significantly higher in basal medium without nitrogen than that determined in the presence of ammonium chloride. Both strains were infective on seedlings of Alnus glutinosa, inducing an approximately similar percentage of nodulated plants (80%), although strain AacIII produced a higher number of nodules per plant (≤15) than strain AacI (≤6). They were also effective for nitrogen fixation in planta, determined by the acetylene reduction assay.

Root nodules ofCeanothus caeruleuscontain both the N2-fixingFrankiaendophyte and a phylogetically related Nod-/Fix-actinomycete

Attempts to isolate the N2-fixing endophyte of Ceanothus caeruleus (Rhamnaceae) root nodules, led to the isolation of nine actinomycetous strains. Owing to their inability to fix nitrogen (Fix-) and nodulate (Nod-), they could not be regarded as the effective endophyte. Characterization was done based on morphological and physiological features and 16S rDNA sequence analysis. The effective Frankia endophyte was characterized without cultivation by amplification, cloning, and sequencing of nearly full length 16S rDNA and partial nifH genes. Phylogenetic analysis based on 16S rDNA revealed that both the effective endophyte and the isolated actinomycetes belong to two different but well-defined lineages within the family Frankiaceae. One lineage is formed mainly by uncultured endophytes that so far have resisted isolation, and the other includes only Fix-/Nod-isolates. Application of temperature gradient gel electrophoresis techniques to actinorhizal nodules allowed us to detect and identify 16S rDNA sequences from both the Fix+and the Fix-nodule inhabitants. Interestingly, these same two sequences were detected on Hippophae rhamnoides nodules obtained after inoculation with Ceanothus caeruleus nodule suspensions. The isolates were located in the outer layers of the nodule.Key words: Frankia, Ceanothus, 16S rDNA, nifH, temperature gradient gel electrophoresis (TGGE), Fix-/Nod-strains.

Frankia in decaying fallen trees devoid of actinorhizal hosts and soil

Fallen trees, recruited by natural catastrophic events, are abundant on the forest floor of many natural forests. They pass through recognizable classes of decomposition, forming a major structural diversity with many ecological functions, and providing habitats for plants, animals, and microorganisms. In greenhouse studies, wood samples collected from within fallen trees decades-old, partially decomposed under both conifer and red alder stands induced effective nodule formation in Alnus rubra, indicating that the fallen trees, devoid of soil and actinorhizal host roots, contained infectious and effective Frankia. Nodule development, growth, and nitrogen fixation were greatest in wood from fallen trees in the alder stand. Frankia is apparently able to live saprophytically or exists in spores in partially decomposed wood.

River and lake sediments as sources of infective Frankia (Alnus)

Several Alnus species are commonly found growing along rivers and lakes. Alnus forms root nodules with N2-fixing Frankia. We used plant bioassays to detect presence of infective Frankia in river and lake sediments from northern Sweden and Alaska. Silty river water and superficial river and lake sediments from all sites contained infective and effective Frankia, but 100-3000 year old lake sediments did not produce nodulated plants. Action by streams, waves and changing water levels transport Frankia in superficial sediments and can provide an inoculum for Alnus roots.

CROSS INOCULATION STUDIES IN Casuarina equisetifolia

An experiment was conducted to identify effective Frankia strain that established good nodulation for higher biomass production in Casuarina equisetifolia through cross inoculation of Frankia strains from four different hosts viz, C. junghuhniana, c. cunninghamiana, Alnus nephalensis and Myrica gale. The result of plant biomass production and nodulation assay revealed that the superior performance of C. Junghuhniana source over other sources tested; on the other hand A. nephalensis and M.gale showed nil performance, but C.cunninghamiana source average performance over control.

Resistance to an ineffective Frankia strain type in Alnus glutinosa (L.) Gaertn.

Characteristics of natural resistance to the ineffective Frankia strain type AgI-WD1 were investigated in different progenies of the host species Alnus glutinosa (L.) Gaertn. with the aid of nodulation experiments. This strain type of Frankia was abundant in the soil of an A. glutinosa stand at Voorne, The Netherlands. Soil suspensions from this site produced high numbers of ineffective root nodules on some of the A. glutinosa seedlings used in biotests, but ii significant number of the seedlings did not produce ineffective nodules. Resistance (R) or susceptibility (S) of the seedlings to Frankia strain type AgI-WD1, as expressed by absence or presence of ineffective root nodules, proved to be persistent in cloned plants of each phenotype. Both R and S-seedlings were susceptible to effective Frankia strains in the soil suspensions. Variation in nodulation time was examined among cloned plants of S-seedlings selected for this trait. Average nodulation times of three selected clones were 11, 26 and 39 d, respectively. Relative growth rates of cloned plants showed no relationship with nodulation time. Both R and S phenotypes were present in all seed-lots of A. glutinosa under study, but the frequency of R phenotypes of seed-lots from different locations ranged from 24 to 100%. R-frequencies of seed-lots of nine trees from a small population of A. glutinosa segregated into two distinct frequency classes which suggested genotypic differentiation of the R-trait among these trees. A significant correlation was found between R-frequencies of seedlots from the same trees over successive years.

The occurrence of Frankia in tropical forest soils of Costa Rica

Infective and effective Frankia were shown to occur in five diverse tropical forest soils of Costa Rica. Results of a plant infection assay indicated that Frankia is a common component of the soil biota in low and high elevation, primary and secondary forest soils. This is the first report of Frankia in lowland tropical rainforests of the Americas. These results suggest either a nonsymbiotic population of soil Frankia, the presence of unknown actinorhizal host species, or an ability of Frankia to be dispersed over long distances.

Characterization of an effective Frankia (ISU 0224887) isolated from nodules of Gymnostoma sumatranum

A Frankia strain ISU 0224887 was isolated from spore negative root nodules of Gymnostoma sumatranum and was grown in pure culture. It was infective and effective for Gymnostoma species but failed to nodulate Allocasuarina and Casuarina seedlings. Light and scanning electron microscopy of it in nitrogen free medium revealed a filamentous mat of septate and branched hyphae bearing sporangia and vesicles capable of fixing nitrogen. The strain also produced an orange pigment after 2 weeks culture. The strain utilized only TWEEN 80 and propionate as sole carbon sources. The different antibiotics used showed varying effects on its growth.

Compatibility ofFrankiaespore positive and spore negative inocula withAlnus glutinosaandAlnus incana

Abstract Growth, nodulation, and nitrogen fixation by Alnus glutinosa (L.) Gaertn. and Alnus incana (L.) Moench inoculated with two Frankia inocula (a spore positive nodule homogenate and a spore negative isolate) were examined. The spore positive inoculum had a very low compatibility with A. glutinosa while a very large compatibility was found with the spore negative strain. Alnus incana had little specificity to the two types of Frankia which allowed the coexistence and the expression of the nitrogen fixation capacity of the two nodule types on the same root system. This result suggests that, despite the competition from indigenous strains, it may be possible to introduce in some areas a highly effective Frankia strain after careful study of the compatibility.

Competitiveness of Frankia strains on Elaeagnus clonal plantlets

Actinorhizae were synthetized on in vitro propagated Elaeagnus angustifolia L. clonal plantlets, by inoculation with combinations of pure cultures of three effective Frankia strains and an ineffective one. Using the OsO4 isolation method, 774 nodule fragments from 79 plantlets were treated and 152 Frankia reisolates were obtained. The soluble protein patterns of 121 reisolates were analyzed by one-dimensional SDS-PAGE, which permitted their positive identification. The protein patterns of all reisolates obtained from plants inoculated with a single strain were indistinguishable from those of the original strain. In six different dual strain inoculations, only one of the two Frankia strains could be reisolated. Thus, some Frankia strains appeared to be more competitive than others toward the Elaeagnus root system.

Competitiveness ofFrankiastrains onElaeagnusclonal plantlets

Actinorhizae were synthetized on in vitro propagated Elaeagnus angustifolia L. clonal plantlets, by inoculation with combinations of pure cultures of three effective Frankia strains and an ineffective one. Using the OsO4 isolation method, 774 nodule fragments from 79 plantlets were treated and 152 Frankia reisolates were obtained. The soluble protein patterns of 121 reisolates were analyzed by one-dimensional SDS-PAGE, which permitted their positive identification. The protein patterns of all reisolates obtained from plants inoculated with a single strain were indistinguishable from those of the original strain. In six different dual strain inoculations, only one of the two Frankia strains could be reisolated. Thus, some Frankia strains appeared to be more competitive than others toward the Elaeagnus root system.