Showing papers on "Frankia published in 2003"

Local and systemic effects of phosphorus and nitrogen on nodulation and nodule function in Alnus incana

Abstract: Phosphorus (P) and nitrogen (N) effects on nodulation, nitrogenase activity and plant growth were studied in the root-hair-infected actinorhizal plant Alnus incana (L.) Moench. A split-root experiment, as well as a short-term experiment with entire root systems and a broader range of P concentrations, showed that P effects were specific on nodulation and not a general stimulation via a plant growth effect. These results indicate that nodule initiation and nodule growth have a high P demand. The split-root assay, comprising seven combinations of two N and two P levels, showed that P could counteract systemic N inhibition of nodulation, but did not counteract N inhibition of nitrogenase activity.

A possible role for phenyl acetic acid (PAA) on Alnus glutinosa nodulation by Frankia

Abstract: Phenylacetic hopanetetrol is a Frankia specific lipid present in vesicles. Phenylacetic acid (PAA) is known as an auxinomimetic, exhibiting the same effect on plant growth as indole acetic acid (IAA). We hypothesize that PAA, only bound by an ester link to the hopanetetrol basic unit, would be easily released and could thus play a role in nodule formation. HPLC and mass spectrometry analysis allowed us to show that 2 Alnus- (ACoN24d and ACN14a) and 2 Elaeagnus-infective strains (EaI1 and EaI3) released PAA into the culture medium, at concentrations of about 10−5 to 10−6 M, whereas IAA was not detected. Furthermore, exogenous PAA added to axenically-grown Alnus glutinosa roots at a concentration of 5 × 10−5 M, resulted in the formation of thick, short lateral roots which resembled actinorhizal nodules. phenylalanine ammonia lyase (PAL) and chalcone syntase (CHS) induction by incompatible and compatible Frankia strains in A. glutinosa roots and the different contents in salicylic acid precursors (cinnamic acid and benzoic acid) observed between nodules and roots support the idea that PAA would be produced in nodules to the detriment of salicylic acid production. These results provide evidence that in actinorhizal root nodules, phenylpropanoid metabolism may play a multiple role in symbiotic interactions including the limitation of the induction of the systematic acquired resistance (SAR) by the plant.

cg12 Expression is specifically linked to infection of root hairs and cortical cells during Casuarina glauca and Allocasuarina verticillata actinorhizal nodule development

Abstract: cg12 is an early actinorhizal nodulin gene from Casuarina glauca encoding a subtilisin-like serine protease. Using transgenic Casuarinaceae plants carrying cg12-gus and cg12-gfp fusions, we have studied the expression pattern conferred by the cg12 promoter region after inoculation with Frankia. cg12 was found to be expressed in root hairs and in root and nodule cortical cells containing Frankia infection threads. cg12 expression was also monitored after inoculation with ineffective Frankia strains, during my-corrhizae formation, and after diverse hormonal treatments. None of these treatments was able to induce its expression, therefore suggesting that cg12 expression is linked to plant cell infection by Frankia strains. Possible roles of cg12 in actinorhizal symbiosis are discussed.

Tripartite associations in an alder: effects of Frankia and Alpova diplophloeus on the growth, nitrogen fixation and mineral acquisition of Alnus tenuifolia

Abstract: The role of tripartite associations among Frankia, Alpova diplophloeus (an ectomycorrhizal fungus) and Alnus tenuifolia in growth, nitrogen fixation, ectomycorrhizal formation, and mineral acquisition of A. tenuifolia was investigated. Seedlings of A. tenuifolia were planted in pots containing a mixture of ground basalt–perlite, or perlite alone, which served as the control. The seedlings were inoculated with Frankia isolated from root nodules of alder, followed by spores of A. diplophloeus and grown for 5 months in a greenhouse. The seedlings grown in the pots with a mixture of ground basalt–perlite after dual inoculation with Frankia and A. diplophloeus had the heaviest shoots and root nodules in dry weight, and showed the greatest nitrogen-fixing ability measured by acetylene reduction. Ectomycorrhizae formed with A. diplophloeus increased when this fungus was inoculated together with Frankia. The mineral composition (P, K, Ca, Fe, Mg, Mn, Na, Si and Al) in the seedlings was also determined. The results of these experiments showed that the tripartite associations could improve the growth, nitrogen fixation and mineral acquisition (rock solubilization) of A. tenuifolia.

Salt tolerance of Casuarina equisetifolia and Frankia Ceq1 strain isolated from the root nodules of C. equisetifolia

Abstract: Effects of NaCl on the seed germination and growth of Casuarina equisetifolia seedlings and multiplication of the Frankia Ceq1 strain isolated from the root nodules of C equisetifolia were examined The germination rate of the seeds markedly decreased as the NaCl concentration increased and germination did not occur at 300 mM NaCl The fresh weight of both shoots and roots of the seedlings treated with NaCl for 6 weeks apparently decreased as the NaCl concentration increased However, root nodules were formed by inoculation with the Frankia Ceq1 strain in some seedlings treated with 300 mM NaCl and the viability of the seedlings at 500 mM NaCl was almost the same as that of the seedlings not subjected to the NaCl treatment The Na+ concentration in the shoots sharply increased with the elevation of the NaCl concentration in the ambient solution, but the level was approximately 300 mM even in the seedlings treated with 500 mM NaCl for 6 weeks On the other hand, the increase of the Na+ concentrat

Actinorhizal nitrogen fixing nodules: infection process, molecular biology and genomics

Abstract: Actinorhizal hosts are non-leguminous perennial plants belonging to 8 angiosperm families. They are capable of forming root nodules as a result of infection by a nitrogen-fixing actinomycete called Frankia. Actinorhizal nodules consist of multiple lobes, each of which represents a modified lateral root with infected cells in the expanded cortex. This article summarizes the most recent knowledge about this original symbiotic process. The infection process is described both at cytological and molecular levels. The use of transgenic Casuarinaceae for studying in actinorhizal nodules the regulation of several symbiotic promoters from legumes is also discussed. With progress in plant genome sequencing, comparative genomics in legumes and actinorhizal plants should contribute to the understanding of the evolutionary history of nitrogen-fixing symbioses. Key words : Nitrogen-fixation, actinorhizal nodules, Frankia, Casuarina, symbiotic gene. African Journal of Biotechnology Vol. 2 (12), pp. 528-538, December 2003

Germination and physiological properties of Frankia spores

Abstract: Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.

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

Abstract: 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.

Distinct patterns of symbiosis-related gene expression in actinorhizal nodules from different plant families.

Abstract: Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia. In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes. In parallel, the phylogeny of actinorhizal plants was examined more closely. The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships. In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).

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

Abstract: 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.

Progress on the genetics of the N2-fixing actinorhizal symbiont Frankia

Abstract: The actinomycete Frankia is of fundamental and ecological interests for several reasons including its wide distribution, its ability to fix nitrogen, differentiate into sporangium and vesicle (specialized cell for nitrogen-fixation), and to nodulate plants from about 24 genera. Here, we present a review on the genetics performed so far on Frankia. At the end of July 2001, 293 kbp of Frankia DNA sequences were found in the databases. Thirty five percent of these sequences corresponded to full gene or gene cluster sequences. These genes could be divided according to their role into 6 key activities: gene translation (rrnA and tRNA pro gene), proteolysis (per genes), assimilation of ammonium (glnA and glnII), protection against superoxide ions (sodF), nitrogen fixation (nif cluster), and plasmid replication. We present a review of these genetic islands; their function, expression, localization and particular properties are discussed. A comparative analysis of Frankia nif genes from various strains and species is presented. An improved nomenclature for some of these genes is suggested to avoid conflicts. Frankia plasmids DNA sequences are also presented. The novel trends in Frankia genetics are described.

The regulation of nodulation, nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

Abstract: N2-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N2 fixation.

A novel calcimycin antibiotic from Gram-positive actinomycete Frankia microsymbiont

Abstract: Frankia as a nitrogen fixing, symbiotic Gram-positive actinomycete has been a subject of tremendous importance in the last few decades. The actinomycete Frankia has been found to be associated with root nodules of most actinorhizal plants (over 200 species in 25 genera been established) (Figure 1 a). With the development of molecular biology, attempts to elucidate the molecular phylogeny and establishment of genetic diversity of the strains have been successful (Figure 1 b). However, since the first record of Frankia isolation from root nodules of Comptonia peregrina, very few attempts were made to understand the antagonistic effects of Frankia on other microbes. Microbial antagonism by a particular species can be a result of competition for nutrients and struggle for survival by the secretion of antagonistic compounds detrimental to the growth of other competing species. Production of antimicrobial compounds in vitro is a wellreported property of numerous microbial strains including actinomycetes. Many actinomycete species including Streptomyces, in particular, are known to produce a vast number of structurally diverse compounds that inhibit the development of other microbes. Actinomycetes are also considered to be a source of commercially valuable bioactive compounds. Production of hydrolysing enzymes, indoles, iron chelating siderophores, and benzonaphthacene quinone metabolites have been reported in Frankia. These antimicrobial metabolites are thought to facilitate Frankia to survive under non-symbiotic conditions. Some studies have shown that Frankia have the potential to inhibit growth of competing soil microbes by producing antimicrobial compounds. Attempts to explicate the antibiotic resistance patterns in Frankia strains have been successful. Frankia strains isolated from different Casuarina sp. have been observed to produce metabolites in culture broths that expressed bioactivity against Gram-negative Pseudomonas solanacearum and Gram-positive Brevibacillus laterosporous. During attempts to isolate pure cultures of Frankia strains, several strains have been found to synthesize yellow, orange, pink or red pigments characterized to be benzonaphthacene quinones that have shown to inhibit the growth of Gram-positive Arthrobacter globiformis, the yeast Candida lipolytica and the deuteromycete Fusarium decemcellulare. Recently, Haansuu et al. screened 39 Frankia strains for observing antimicrobial and calcium antagonistic activities; where one of the strains obtained was found to synthesize coloured pigments in growth medium, regarded to be commonly present and ubiquitously expressed in Frankia. The chemical nature of these unknown compounds was determined to observe antimicrobial activities. Structural elucidation of the antimicrobial compounds isolated from Frankia strains AiPs1 and AiPs3 (isolates from Alnus incana and Pinus sylvestris) has been reassigned to the calcimycin class of antibiotics and pyrrolether ionophores. Earlier, initial structural elucidation of the compounds derived from Nuclear Magnetic Resonance (NMR)

Choosing a reporter for gene expression studies in transgenic actinorhizal plants of the Casuarinaceae family

Abstract: Transgenic Casuarinaceae and reporter genes provide valuable tools to study gene expression in transgenic actinorhizal nodules. In this paper, we discuss the use of s-glucuronidase for the histochemical localization and quantification of gene expression in transgenic plants of Allocasuarina verticillata and Casuarina glauca nodulated by the actinomycete Frankia. We also report on the genetic transformation of A. verticillata by the Agrobacterium tumefaciens strain C58C1(pGV2260) containing the 35S-mgfp5-ER construct encoding a modified green fluorescent protein of Aequorea victoria in a binary vector. The evolution of the GFP fluorescence was monitored through all stages of the regeneration process. The data indicate that GFP is not toxic in Casuarinaceae and that this reporter gene can be used for visual screening of transformed calli and transgenic plants. The fluorescence pattern of gfp provides a new tool for monitoring in vivo transgenc expression in actinorhizal plants.

Ammonium assimilation in root nodules of actinorhizal Discaria trinervis . Regulation of enzyme activities and protein levels by the availability of macronutrients (N, P and C)

Abstract: Asparagine was found to be the main N compound exported from Discaria trinervis nodules. Aspartate (Asp), glutamate (Glu), alanine (Ala) and serine (Ser) were also detected in root xylem sap, but at lower concentrations. A comparable picture is found in nodulated alfalfa. We hypothesized that a similar set of enzymes for Asn synthesis was present in D. trinervis nodules. We demonstrate the expression of most of the enzymes involved in the synthesis of Asn from NH 4 + and oxoacids, in nodules — but not in roots — of fully symbiotic D. trinervis. By complementation of enzyme assays (A) and immunodetection (I) we detected glutamane-synthetase (GSA.I), Asp-aminotransferase (AATA), malate-dehydrogenase (MDHA.I, at least two isoforms), Glu-dehydrogenase (GDHA), Glu-synthase (GOGATI) and Asn-synthetase (ASI). PEP-carboxylase (PEPC) activity was not detected. We previously shown that N acts as a negative regulator of nodulation and nodule growth, while P is a strong stimulator for nodule growth. We present data on the regulation of nodule N metabolism by altering, during 4 weeks, the availability of N, P and light in symbiotic D. trinervis. NH4NO3 (2 mM) induced inactivation and degradation of nodule GS, MDH and AS, but activation of GDH and AAT; the amount of nitrogenase components was not affected. A 10-fold increase in P supply did not greatly affect activity and amount of enzymes, suggesting that N metabolism is not P-limited in nodules. On the other hand, suppression of P supply induced an important reduction of nodule GS, GOGAT, MDH and AS protein levels, although nitrogenase was not affected. GDH was the only measured activity that was stimulated by limiting P supply. Shading plants did result in complete degradation of nitrogenase and partial degradation of GS, AS and nodule-specific MDH isoform, but GDH and AAT were activated. These results are discussed in connection with the regulation of nodulation and nodule growth in D. trinervis.

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

Abstract: 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.

Effects of land irrigation with partially-treated wastewater on Frankia survival and infectivity

Abstract: The effect of partially treated wastewater on Frankia growth and infectivity on Casuarina equisetifolia plants was studied. This water contained high amounts of cyanide, arsenic, mercury ions, and contaminating microorganisms. Three Frankia strains were grown on elevated concentrations of wastewater up to 100% as a preliminary test to determine the possibility of their growth on these concentrations. Their growth was reduced to about 50% with slight variations between strains. Twelve Frankia strains were then grown on 100% wastewater and their growth was not much affected but showed only variations in growth rates. Six of these strains, when grown on wastewater, lost their infectivity when inoculated on Casuarina seedlings irrigated with the same water. In contrast, nodulation occurred on plants that were irrigated with wastewater when inoculated with Frankia grown on liquid BuCT medium although nodule size and weight was reduced for all strains. Statistical analysis (two-way ANOVA) showed no significant differences (P = 0.05) in the number of nodules or shoot dry weight for different treatments. Significant differences were found between treatments in root length, shoot height and root and nodule dry weight. No significant differences were recorded also within the same treatments for all plant growth criteria. These results indicate the possibility of inhibitory effect on the infectivity of natural Frankia populations but not on the selected and introduced (i.e., tolerant) strains.

Infectivity and effectivity of Frankia strains from the Rhamnaceae family on different actinorhizal plants

Abstract: Ten strains of Frankia isolated from root nodules of plant species from five genera of the host family Rhamnaceae were assayed in cross inoculation assays. They were tested on host plants belonging to four actinorhizal families: Trevoa trinervis (Rhamnaceae), Elaeagnus angustifolia (Elaeagnaceae), Alnus glutinosa (Betulaceae) and Casuarina cunninghamiana (Casuarinaceae). All Frankia strains from the Rhamnaceae were able to infect and nodulate both T. trinervis and E. angustifolia. Strain ChI4 isolated from Colletia hystrix was also infective on Alnus glutinosa. All nodules showed a positive acetylene reduction indicating that the microsymbionts used as inoculants were effective in nitrogen fixation. The results suggest that Frankia strains from Rhamnaceae belong to the Elaeagnus-infective subdivision of the genus Frankia.

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

Abstract: 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.

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

Abstract: 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 sieb...

A Type-A chalcone isomerase mRNA is highly expressed in the root nodules ofElaeagnus umbellate

Abstract: We have used the hybridization-competition method to isolateEuNOD-CHI from a root nodule cDNA library ofElaeagnus umbellate. This cDNA clone encodes chalcone isomerase (CHI) for a protein of 256 amino-acid residues and a mature molecular mass of 28 kDa. Multiple sequence alignment and phylogenetic analysis have demonstrated that EuNOD-CHI can be classified as Type I. Moreover, northern hybridization shows that theEuNOD-CHI gene is highly expressed in root nodules, with levels increasing during nodule development The highest level of expression is at 6 to 8 weeks after inoculation, decreasing thereafter. Genomic Southern hybridization also demonstrates thatEuNOD-CHI has as many as two copies in theE umbellate genome. Taken together with the previous results, we propose that the higher expression level of theEuNOD-CHI gene in root nodules is likely associated with this species’ defense mechanism against infection byFrankia.

Dual Inoculation Increases Plant Growth with Frankia on Red Alder (Alnus rubra Bong.) in Fumigated Nursery Beds

Abstract: Inoculation trials for red alder (Alnus rubra Bong.) bare-root seedling production were set up in fumigated nursery beds. Frankia inoculum was applied in a peat mix carrier, either as a single strain (strains ArI5 or AvcI1) or as a combination of the two strains at the same total rate of application. The plots were laid out in four blocks of four treatments: control, strain ArI5, strain AvcI1, and dual inocula. Plots were 61 cm by 122 cm with 15-cm buffer strips. Number, height, and percentage nodulation of the seedlings were determined at mid-season. At lifting, seedlings were counted and sorted into size-classes for subsampling. Number, size, dry weight, and degree of nodulation were determined on this stratified sample. Frankia DNA from nodules was analyzed by DNA fingerprinting. The controls were poorly nodulated and grew slowly. The single strain inoculum for ArI5 produced larger seedlings than for AvcI1, both at mid-season and at lifting. The dual-inocula treatment produced larger seedlings than all other treatments. The AvcI1 single-inoculum nodules primarily produced fingerprints identical to AvcI1, but nodule fingerprints from all other treatments were dominated by ArI5 patterns. Thus, the observed seedling growth advantage with dual inocula was not attributable to nodule occupancy differences.

Nitrogenase activity and root nodule metabolism in response to O2 and short-term N2 deprivation in dark-treated Frankia-Alnus incana plants

Abstract: Inhibition of nitrogenase (EC 1.18.6.1) activity by O2 has been suggested to be an early response to disturbance in carbon supply to root nodules in the Frankia-Alnus incana symbiosis. Intact nodulated root systems of plants kept in prolonged darkness of 22 h were used to test responses to O2 and short-term N2 deprivation (1 h in Ar:O2). By using a Frankia lacking uptake hydrogenase it was possible to follow nitrogenase activity over time as H2 evolution in a gas exchange system. Respiration was simultaneously recorded as CO2 evolution. Dark-treated plants had lower initial nitrogenase activity in N2:O2 (68% of controls), which declined further during a 1-h period in the assay system in N2:O2 at 21 and 17% O2, but not at 13% O2. When dark-treated plants were deprived of N2 at 21 and 17% O2 nitrogenase activity declined rapidly to 61 and 74%, respectively, after 20 min, compared with control plants continuously kept in their normal light regime. In contrast, there was no decline in dark-treated plants at 13% O2, and only a smaller and temporary decline in control plants at 21% O2. When dark-treated plants were kept at 21% O2 during 45 min prior to N2 deprivation at 17% O2 the decline was abolished. This supports the idea that the decline in nitrogenase activity observed in N2:O2 at 21% O2 and during N2 deprivation was caused by O2, which affected a sensitive nodule fraction. Nodule contents of the amino acids Gln and Cit decreased during N2 deprivation, suggesting decreased assimilation of NH4+. Contents of ATP and ADP in nodules were not affected by short-term N2 deprivation. ATP/ADP ratios were about 5 indicating a highly aerobic metabolism in the root nodule. We conclude that nitrogenase activity of Alnus plants exposed to prolonged darkness becomes more sensitive to inactivation by O2. It seemed that dark-treated plants could not adjust their nodule metabolism at higher perceived pO2 and during cessation of NH4+ production.

Molecular cloning and complementation analysis of nifV gene from Frankia EuIK1 strain.

Abstract: The nifV gene from the Frankia EuIK1 strain, a symbiont of Elaeagnus umbellata, was cloned and a complementation test using the Klebsiella pneumoniae nifV mutant was performed to verify its function. The nifV ORF consists of 1245 bp, which encodes 414 amino acids. However, the putative promoter and Shine-Dalgarno sequences were not found in the 5' region of the ORF. The Frankia EuIK1 nifV ORF showed about a 70% nucleotide identity and 80% amino acid similarity with that of Frankia sp. FaC1. In the upstream region of the nifV, a putative ORF that showed a 51% nucleotide identity with the afcD gene from Burkholderia cepacia BC11 was found. The other partial ORF that showed a 59% identity with the pkaD gene from Streptomyces coelicolor A(3) was found in the downstream region. In this respect, Frankia EuIK1 nifV has an unusual location on the genome, considering the nif gene organization. A phylogenetic analysis revealed that the NifV from Frankia EuIK1 was close to those from two Alnus-infective Frankia species, and they were grouped with those of the alpha-class proteobacteria, supporting the vertical descent of nifV. The transcription and function of Frankia EuIK1 nifV were verified by a RT-PCR analysis and complementation test with the K. pneumoniae mutant, respectively. These results suggested that Frankia EuIK1 nifV is a functional gene.

Genomic fingerprinting of Frankia strains by PCR-based techniques. Assessment of a primer based on the sequence of 16S rRNA gene of Escherichia coli

Abstract: Polimerase Chain Reaction (PCR)-based genomic fingerprints of 16 Frankia isolates were obtained using two different primers. rep-PCR DNA fingerprints were obtained by using DR1R primer, and Randomly Amplified Polymorphic DNA (RAPD) fingerprints by using a large primer derived from the 16S rDNA sequence of Escherichia coli (879F primer). According to the results obtained, primer DR1R generates strain-specific patterns. However, primer 879F yielded an identical band patterns in two Frankia strains, CcI3 and UGL 020603, isolated from different Casuarina species and geographical origins, indicating that it could identify genomic fingerprints at a higher taxonomic level (subspecies or species) than DR1R primer. To verify this hypothesis, we tested the primer 879F with eight strains of Clavibacter michiganensis, another actinobacterium with high G+C content, which includes several well-defined subspecies. Primer 879F identified a unique band pattern in all strains within the same subspecies but different patterns for other subspecies, indicating that it generates subspecies-specific genomic fingerprints. Consequently, Frankia Cc13 and Frankia UGL 020603 should be included in the same subspecies and the remaining Frankia strains used in this study in different subspecies. An UPGMA dendrogram of the 879F-PCR fingerprint patterns shows that the Frankia strains used in this study could be clustered into groups that broadly reflects the host plants from which they were derived. These results are consistent with those obtained by other authors using different techniques. Because primer 879F yields genomic fingerprints at taxonomic level upper than strain level, it may be a valuable tool in the polyphasic approach to Frankia taxonomy, where other classical taxonomic methods are barely applicable. Taking into account that several primers can be designed from ribosomal sequences in order to obtain RAPD patterns, we propose to name these methods according to the position of such primers in the 16S rRNA sequences of Escherichia coli. In this way, we name 879F-RAPD fingerprinting the procedure used in this study.

Improving dispersed growth of Frankia using Carbopol

Abstract: In laboratory culture, strains of the actinomycete Frankia grow slowly and form dense mycelial pellets. In this study, we have shown that by adding the anionic polymer Carbopol 941® to liquid growth media, dispersed growth of Frankia is improved. Cell protein measurements indicated higher biomass production in cultures maintained in media supplemented with Carbopol. Fluorescence staining and microscopy were used to compare viability of well-dispersed mycelia and pellets.

Evolutionary implications of nucleotide sequence relatedness between Alnus nepalensis and Alnus glutinosa and also between corresponding Frankia microsymbionts

Abstract: Frankia DNAs were isolated directly from root nodules of Alnus nepalensis and Alnus nitida collected from various natural sites in India. For comparison, a nodule sample from Alnus glutinosa was also collected from Tuebingen, Germany. Nucleotide sequence analyses of amplified 16S–23S ITS region revealed that one of the microsymbionts from Alnus nepalensis was closely related to the microsymbiont from Alnus glutinosa. A similar exercise on the host was also carried out. It was found that one sample of Alnus nepalensis was closely related to Alnus glutinosa sequence from Europe. Since both Frankia and the host sequences studied revealed proximity between Alnus glutinosa and Alnus nepalensis, it is hypothesised that the common progenitor of all the alders first entered into an association with Frankia, and the symbiotic association has evolved since.

Expression pattern of ara12*, an Arabidopsis homologue of the nodule-specific actinorhizal subtilases cg12/ag12

Abstract: In response to infection by Frankia, cells of Alma glutinosa and Casuarina glauca nodules strongly express a subtilase-gene (ag12 and cg12 respectively). In order to gain a better understanding of the function of these genes in the symbiotic process, we have analysed the gene expression of ara12, an Arabidopsis thaliana homologue of cg12 and ag12. Transcription fusion of the promoter region of ara 12 to the β-glucuronidase gene was introduced into A. thaliana. Para12-gus expression was found in young developing tissues. This result suggests that ara 12 might be involved in proteins or polypeptides processing during A. thaliana development. We therefore speculate that cg12 and agl2, the actinorhizal homologues of ara12 might be involved in differentiation of Frankia infected cells in nodules.