Discaria Trinervis Research Articles

Cell remodeling and subtilase gene expression in the actinorhizal plant Discaria trinervis highlight host orchestration of intercellular Frankia colonization.

Nitrogen-fixing filamentous Frankia colonize the root tissues of its actinorhizal host Discaria trinervis via an exclusively intercellular pathway. Here we present studies aimed at uncovering mechanisms associated with this little-researched mode of root entry, and in particular the extent to which the host plant is an active partner during this process. Detailed characterization of the expression patterns of infection-associated actinorhizal host genes has provided valuable tools to identify intercellular infection sites, thus allowing invivo confocal microscopic studies of the early stages of Frankia colonization. The subtilisin-like serine protease gene Dt12, as well as its Casuarina glauca homolog Cg12, are specifically expressed at sites of Frankia intercellular colonization of D.trinervis outer root tissues. This is accompanied by nucleo-cytoplasmic reorganization in the adjacent host cells and major remodeling of the intercellular apoplastic compartment. These findings lead us to propose that the actinorhizal host plays a major role in modifying both the size and composition of the intercellular apoplast in order to accommodate the filamentous microsymbiont. The implications of these findings are discussed in the light of the analogies that can be made with the orchestrating role of host legumes during intracellular root hair colonization by nitrogen-fixing rhizobia.

Frankia discariae sp. nov.: an infective and effective microsymbiont isolated from the root nodule of Discaria trinervis.

Strain BCU110501T was the first isolate reported to fulfill Koch's postulates by inducing effective nodules on its host plant of origin Discaria trinervis (Rhalmnaceae). Based on 16S rRNA gene sequence similarities, the strain was found to be most closely related to the type strain of Frankia elaeagni DSM 46783T (98.6%) followed by F. alni DSM 45986T (98.2%), F. casuarinae DSM 45818T (97.8%) and F. inefficacies DSM 45817T (97.8%). Digital DNA:DNA hybridizations (dDDH) between strain BCU110501Tand the type strains of other Frankia species were clearly below the cutoff point of 70%. The G+C content of DNA is 72.36%. The cell wall of strain BCU110501T contained meso-diaminopimelic acid and the cell sugars were galactose, glucose, mannose, xylose and ribose. Polar lipids were phosphatidylinositol (PI), diphosphatidylglycerol (DPG), glycophospholipid (GPL1-3), phosphatidylglycerol (PG) and an unknown lipid (L). The major fatty acids of strain BCU110501T consisted of iso-C16:0, C17:1 w8c and C16:0. Major menaquinones were MK9 (H4), MK9 (H6) and MK9 (H2). Based on these analyses, strain BCU110501T (=DSM 46785T=CECT 9042T) should be classified as the type strain of a novel Frankia species, for which the name Frankia discariae sp. nov. is proposed.

Interactions between Frankia BCU110501 (actinorhiza) and Gigaspora rosea (arbuscular mycorrhiza) with Discaria trinervis studied by spot inoculation

Root endosymbioses are associations between plants and soil microorganisms which contribute worldwide to plant nutrition and fitness. The most common associations is with arbuscular mycorrhizal fungi (AMF) and this is formed by the majority of land plants with fungi belonging to the Glomeromycota. Another important association is that formed by the Gram-positive actinobacteria, Frankia, and actinorhizal plants such as Discaria trinervis. In this paper we studied the effect of dual symbioses resulting from spot inoculation. This method was developed and tested to ensure actinorhizal nodule development only at the site of inoculation. Our results show that both symbioses can coexist and that AMF and Frankia can stimulate plant growth both as a single inoculum and in combination. Moreover, we suggest that there is no interference between the regulation of actinorhizal and arbuscular mycorrhizal symbiosis in D. trinervis, and that there is a synergistic effect on plant growth following the development of both symbioses.

Role of auxin during intercellular infection of Discaria trinervis by Frankia.

Nitrogen-fixing nodules induced by Frankia in the actinorhizal plant Discaria trinervis result from a primitive intercellular root invasion pathway that does not involve root hair deformation and infection threads. Here, we analyzed the role of auxin in this intercellular infection pathway at the molecular level and compared it with our previous work in the intracellular infected actinorhizal plant Casuarina glauca. Immunolocalisation experiments showed that auxin accumulated in Frankia-infected cells in both systems. We then characterized the expression of auxin transporters in D. trinervis nodules. No activation of the heterologous CgAUX1 promoter was detected in infected cells in D. trinervis. These results were confirmed with the endogenous D. trinervis gene, DtAUX1. However, DtAUX1 was expressed in the nodule meristem. Consistently, transgenic D. trinervis plants containing the auxin response marker DR5:VENUS showed expression of the reporter gene in the meristem. Immunolocalisation experiments using an antibody against the auxin efflux carrier PIN1, revealed the presence of this transporter in the plasma membrane of infected cells. Finally, we used in silico cellular models to analyse auxin fluxes in D. trinervis nodules. Our results point to the existence of divergent roles of auxin in intercellularly- and intracellularly-infected actinorhizal plants, an ancestral infection pathways leading to root nodule symbioses.

The independent acquisition of plant root nitrogen-fixing symbiosis in Fabids recruited the same genetic pathway for nodule organogenesis.

Only species belonging to the Fabid clade, limited to four classes and ten families of Angiosperms, are able to form nitrogen-fixing root nodule symbioses (RNS) with soil bacteria. This concerns plants of the legume family (Fabaceae) and Parasponia (Cannabaceae) associated with the Gram-negative proteobacteria collectively called rhizobia and actinorhizal plants associated with the Gram-positive actinomycetes of the genus Frankia. Calcium and calmodulin-dependent protein kinase (CCaMK) is a key component of the common signaling pathway leading to both rhizobial and arbuscular mycorrhizal symbioses (AM) and plays a central role in cross-signaling between root nodule organogenesis and infection processes. Here, we show that CCaMK is also needed for successful actinorhiza formation and interaction with AM fungi in the actinorhizal tree Casuarina glauca and is also able to restore both nodulation and AM symbioses in a Medicago truncatula ccamk mutant. Besides, we expressed auto-active CgCCaMK lacking the auto-inhibitory/CaM domain in two actinorhizal species: C. glauca (Casuarinaceae), which develops an intracellular infection pathway, and Discaria trinervis (Rhamnaceae) which is characterized by an ancestral intercellular infection mechanism. In both species, we found induction of nodulation independent of Frankia similar to response to the activation of CCaMK in the rhizobia-legume symbiosis and conclude that the regulation of actinorhiza organogenesis is conserved regardless of the infection mode. It has been suggested that rhizobial and actinorhizal symbioses originated from a common ancestor with several independent evolutionary origins. Our findings are consistent with the recruitment of a similar genetic pathway governing rhizobial and Frankia nodule organogenesis.

Vertebrate faeces as sources of nodulating Frankia in Patagonia

Frankia strains nodulate the native actinorhizal plant Ochetophila trinervis (sin. Discaria trinervis), which grows in stream margins and nearby areas in northwest Patagonia (Argentina). Infective Frankia are found in soils with presence of host plants but also may be found in areas lacking them. This may be partly explained by water transport of Frankia propagules but there are other possible sources. The aim of this study was to discover whether the faeces of introduced mammalian herbivores, including cows (Bos taurus, adult and calf), horses (Equus caballus), sheep (Ovis aries), red and/or fallow deer (Cervus elaphus and Dama dama, respectively), wild boar (Sus scrofa), European hare (Lepus capensis), or the native upland goose (Chloephaga picta), could be a source of infective Frankia, and enhance its dispersal. Faecal material and soil samples were aseptically sampled in different plant communities, and tested via plant bioassays using O. trinervis. The faeces of all animals contained infective Frankia and led to an effective symbiosis with this plant. Faeces of large introduced herbivores gave rise to higher nodulation (number of nodulated plants with respect to the total number of inoculated plants) than faeces of hare and upland goose. Soils from the sites where the cow (two sites), sheep, wild boar and deer faeces were collected did not contain infective Frankia. This suggests that the animals may have ingested Frankia from plant material and that the Frankia propagules passed through the digestive tracts of the animals without losing its infectivity. We conclude that the faeces of large introduced herbivores contribute to the dispersal of infective Frankia in Northwest Patagonia.

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.

Composite Actinorhizal Plants with Transgenic Roots for the Study of Symbiotic Associations with Frankia

More than 200 species of dicotyledonous plants belonging to eight different families and 24 genera can establish actinorhizal symbiosis with the nitrogen-fixing soil actinomycete Frankia. Compared to the symbiotic interaction between legumes and rhizobia, little is known about the molecular basis of the infection process and nodule formation in actinorhizal plants. Here, we review a gene transfer system based on Agrobacterium rhizogenes that opens the possibility to rapidly analyze the function of candidate symbiotic genes. The transformation protocol generates “composite plants” that consist of a nontransgenic aerial part with transformed hairy roots. Composite plants have already been obtained in three different species of actinorhizal plants, including the tropical tree species Casuarina glauca, the Patagonian shrub Discaria trinervis, and the nonwoody plant Datisca glomerata. The potential of this technique to advancing our understanding of the molecular mechanisms underlying infection by Frankia is demonstrated by functional analyses of symbiotic genes.

Transformed Hairy Roots of the actinorhizal shrub Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection

Background Nitrogen is a major limiting factor of plant growth in many ecosystems. Root nodule symbiosis (RNS) is one of the most efficient adaptations allowing plants to cope with nitrogen deficiency by establishing a symbiotic association with diazotrophic bacteria able to produce ammonium from atmospheric nitrogen. Nevertheless RNS is restricted to two groups of plants: legumes and Parasponia (Celtidaceae), that interact with a group of gram-negative proteobacteria collectively called rhizobia, and actinorhizal plants, a group of 220 species, mostly shrubs and trees distributed in the orders Fagales, Cucurbitales and Rosales, that interact with gram-positive actinomycetes of the genus Frankia[1]. All these plants belong to the Rosid I clade, suggesting a common origin for the ability to establish RNS [2]. In recent decades a strong research effort focused on model legumes lead to the identification of key molecular actors involved in nodulation, including the bacterial signalling molecules, the Nod factors and several genes involved in the symbiotic signalling pathways [3]. Much less is known in non model legumes and actinorhizal plants, particularly in species that are not infected like model legumes through root hairs but show more ancestral infection mechanisms like crack entry or intercellular infection. Yet important cues regarding the diversity and evolution of RNS are being found precisely in these more primitive non-model systems [4,5]. Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized [6,7]. Intercellular infection has been described in Discaria trinervis, an actinorhizal shrub belonging to the Rosales order [8]. To decipher the molecular mechanisms underlying intercellular infection with Frankia, we set up an efficient genetic transformation protocol for D. trinervis based on A. rhizogenes.

Reproductive ecology of Ochetophila trinervis in Northwest Patagonia.

Native actinorhizal species Ochetophila trinervis (Gillies ex Hook. & Arn.) Poepp. ex Miers, also known as Discaria trinervis (Kellermann et al. 2005), grows along watercourses and distant from them, along a rainfall gradient in north-west Patagonia. We studied the reproductive ecology of this species under different macro- and micro-environmental conditions, in three zones in a rainfall gradient (western, intermediate and eastern) with two sites for each one, near and distant to a watercourse (riparian and dry-land). We performed field studies and germination trials. Plant size, reproductive effort (seed abundance per branch) and seed bank size of O. trinervis, were favoured by the proximity of streams in the drier environments of the gradient. The abundance of seedlings and saplings in the field was very low, which was in agreement with a lack of germination in the field, despite the good germination capacity of seeds. Sexual reproduction of O. trinervis was affected by the low abundance of seedlings. Lower rainfall, higher temperatures and a longer growing season along the gradient favour the potential regeneration of the species. Because of its nitrogen-fixing capacity and other features, O. trinervis has potential for reclamation of eroded lands in Patagonia. Patterns of seed biology and regeneration presented in this study will aid in the use of O. trinervis in the reclamation of disturbed lands.

Diffusible factors from Frankia modify nodulation kinetics in Discaria trinervis, an intercellular root-infected actinorhizal symbiosis.

Frankia BCU110501 induces nitrogen-fixing root nodules in Discaria trinervis (Gillies ex Hook. & Arn.) Reiche (Rhamnaceae) via intercellular colonisation, without root hair deformation. It produces diffusible factors (DFs) that might be involved in early interactions with the D. trinervis roots, playing a role in the nodulation process. The induction of root nodule development in actinorhizal symbiosis would depend on the concentration of factors produced by the bacteria and the plant. A detailed analysis of nodulation kinetics revealed that these DFs produce changes at the level of initial rate of nodulation and also in nodulation profile. Diluted Frankia BCU110501 inoculum could be activated in less than 96h by DFs produced by Frankia BCU110501 cells that had been previously washed. Biochemical characterisation showed that Frankia BCU110501 DFs have a molecular weight of <12kDa, are negatively charged at pH 7.0 and seem to contain a peptide bond necessary for their activity. Frankia BCU110501, belonging to Frankia Clade 3, does not induce nodules in Alnus acuminata H.B.K. ssp. acuminata but is able to deform root hairs, as do Frankia strains from Clade 1. The root hair deforming activity of Frankia BCU110501 DFs show the same biochemical characteristics of the DFs involved in nodulation of D. trinervis. These results suggest that Frankia symbiotic factors have a basic structure regardless of the infection pathway of the host plant.

Diffusible factors involved in early interactions of actinorhizal symbiosis are modulated by the host plant but are not enough to break the host range barrier.

Nodulation kinetics were analysed in two nitrogen-fixing actinorhizal symbioses that show different pathways for infection: Alnus acuminata H. B. K., which is infected by Frankia ArI3, and Discaria trinervis (Hooker et Arnot) Reiche, which is infected by Frankia BCU110501. Both pairs are incompatible in cross-inoculation experiments. The dose-response effects in nodulation were studied in A. acuminata seedlings using different concentrations of compatible and incompatible bacteria in co-inoculation experiments. Restriction fragment length polymorphism PCR analysis and plant-trapping analysis showed no co-occupation in A. acuminata nodules when plants were co-inoculated with Frankia BCU110501 and Frankia ArI3. Despite the lack of co-occupation, the noninfective BCU110501 could modify the nodulation parameters of the non-host A. acuminata when infective ArI3 was present in the inoculum. The results suggest that although BCU110501 was not able to induce nodulation in A. acuminata, its interaction with the plant could induce autoregulation as if some level of infection or partial recognition could be achieved. We explored the possibility that physiological complementation of the heterologous Frankia BCU110501 for nodulation of A. acuminata originated in the homologous Frankia ArI3 in the presence of compatible root exudates. Despite the possibility of full activation between bacteria and the host, there was no co-infection of Frankia BCU110501 in Alnus or of Frankia ArI3 in Discaria either. These negative results suggest a physical recognition barrier in actinorhizal symbiosis that operates after early interactions, involving something other than root exudates and diffusible factors of bacterial or plant origin, regardless of the infection pathway.

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.

Variation of 15N natural abundance in leaves and nodules of actinorhizal shrubs in Northwest Patagonia

This study was performed to assess the N2-fixing capability of the native actinorhizal species Ochetophila trinervis (sin. Discaria trinervis) and Discaria chacaye (Rhamnaceae) in Northwest Patagonia. We measured the N concentration and 15N natural abundance in leaves and nodules of O. trinervis and D. chacaye, in leaves of associated non-actinorhizal vegetation, and in the soils under each sampled plant. O. trinervis and D. chacaye had foliar N concentrations that were about twice that of non-actinorhizal shrubs growing at the same sites, even though soils varied four-fold in total N across the sites. Leaves of both actinorhizal plants had a similar δ15N at any site and were close to atmospheric values. The foliar δ15N of non-actinorhizal plants and soil δ15N were strongly correlated across the sites. Nodules were depleted in δ15N relative to the foliage of the respective actinorhizal species. In conjunction with the uniformly high foliage N concentration of these actinorhizal plants and the universal presence of vesicles observed in root nodules, these data strongly suggest that O. trinervis and D. chacaye obtain a significant amount of their N from N2 fixation. To calculate the proportion of N derived from atmosphere, theoretical B-values were estimated. In all cases where the δ15N of fixing and reference foliage were significantly different, O. trinervis and D. chacaye obtained almost all of their N from N2 fixation. These results are the first to demonstrate N2 fixation by O. trinervis and D. chacaye in the field and therefore suggest an important role for these actinorhizal plants in the N economy of ecosystems in northwest Patagonia as well as their potential use for restoration of degraded lands in this region.

Effects of calcium in the nitrogen-fixing symbiosis between actinorhizal Discaria trinervis (Rhamnaceae) and Frankia

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.

Saprophytic Actinomycetes Promote Nodulation in Medicago sativa-Sinorhizobium meliloti Symbiosis in the Presence of High N

Saprophytic rhizoactinomycetes isolated from the root nodule surface of the nitrogen-fixing actinorhizal plant Discaria trinervis, Streptomyces MM40, Actinoplanes ME3, and Micromonospora MM18, previously shown to stimulate nodulation in Frankia-Discaria trinervis symbiosis, were assayed as co-inoculants with Sinorhizobium meliloti 2011 on Medicago sativa. When plants were fertilized with a low level of N (0.07 mM), the inoculation of the actinomycetes alone did not show any effect on plant growth. Meanwhile, when actinomycetes were co-inoculated with S. meliloti, nodulation and plant growth were significantly stimulated compared to plants inoculated with only S. meliloti. The analysis of nodulation kinetics of simultaneously or delayed co-inoculations suggests that the effect of the actinomycetes operates in early infection and nodule development counteracting the autoregulation of nodulation by the plant. Because the actinomycete effect was found in the symbiotic nitrogen-fixing state of the plant, we investigated the effects of the actinomycetes, in single inoculation or co-inoculation with S. meliloti, on plants grown under a high level of N (7 mM) that was inhibitory for nodulation by S. meliloti. The inoculation of the actinomycetes alone did not show any effect on plant growth although high N was available. Unexpectedly, the co-inoculation of actinomycetes with S. meliloti on plants grown with high N (7 mM) significantly stimulates nodulation, clearly counteracting the inhibition of nodulation by high N. These results corroborate that the interaction of rhizoactinomycetes would interfere with the autoregulation of nodulation in alfalfa mediated by high N, opening new research lines of potential agronomical applications.

Analysis of nodulation kinetics in Frankia–Discaria trinervis symbiosis reveals different factors involved in the nodulation process

The induction of root nodule development in actinorhizal symbiosis would depend on the concentration of factors produced by the bacteria and the plant. A detailed analysis of nodulation description parameters revealed different factors related to the nodulation process. The initial time for nodulation (t(0)), the initial nodulation rate (v(0)) and the total time of nodule development (t(NOD)) were defined and consequently quantified in different experimental conditions: co-inoculation of Discaria trinervis with increasing concentrations of different non-infective bacteria together with the full compatible infective Frankia strain (the indicator strain) used at a limiting concentration or by changing plant factor(s) concentration. All the above nodulation parameters were modified by changing doses of full compatibility infective strain Frankia BCU110501; v(0) appears to be an expression of symbiotic recognition between partners as only fully symbiotic indicator Frankia BCU110501 was able to change it; t(0) seems not to reflect symbiotic recognition because it can also be modified by non-infective Frankia but suggest the existence of a basic level of plant microbe recognition. The initial time for nodulation t(0), reflecting the time required for the early interactions toward nodulation, is an inverse measure of the ability to establish early interactions toward nodulation. The increase in plant factors concentration also reduces t(0) values, suggesting that a plant factor is involved and favors very early interactions. Increases in plant factors concentration also modify the final number of nodules per plant and the nodule cluster profile along the taproot as an expression of the autoregulation phenomenon. Meanwhile, Frankia inoculums' concentration, either infective or not, modified t(NOD) in an opposite way plant factors did. In conclusion, the analysis of nodulation kinetics appears to be an appropriate tool to investigate factors involved in the symbiotic interaction leading to the formation of nitrogen-fixing nodules.

Discaria trinervis – Frankia symbiosis promotion by saprophytic actinomycetes

The influence of saprophytic actinomycetes strains on the Discaria trinervis - Frankia actinorhizal symbiosis was investigated. Three strains out of 122 isolated from the rhizosphere and rhizoplane of D. trinervis with multiple enzymatic activities, were selected for plant growth experiments: Streptomyces (BCRU-MM40), Actinoplanes (BCRU-ME3) and Micromonospora (BCRU-MM18). Inoculated seedlings of Discaria trinervis were grown in glass tubes with vermiculite-sand for 12 weeks. They were inoculated either with a single saprophytic strain or a combination of one or two of them together with the symbiotic N(2) fixing strain Frankia BCU110501. The saprophytic strains were applied in two experimental series, i.e. mycelium + supernatant simultaneously or mycelium and supernatant (growth medium free of cells) separately. Micromonospora strain MM18 showed a direct promotion effect on shoot growth, when plants were inoculated with mycelium and supernatant together. Streptomyces strain MM40 and Actinoplanes strain ME3 promoted the actinorhizal symbiosis with Frankia and consequently the development of plant shoots, when supernatant was involved as inoculum. It is supposed, that the strains MM18, MM40 and ME3 produce bioactive metabolites, which are released into the culture medium. The saprophytic strains studied could be considered as "promoting or helper rhizoactinomycetes" of the actinorhizal plant D. trinervis.

Cover Picture: J. Basic Microbiol. 3/2007

AbstractActinobacteria of the genus Frankia are able to form a symbiosis with plant roots able to fix nitrogen. The actinorhizal plant Discaria trinervis was co‐inoculated with Frankia ‐strain BCU 110501 and in additon with a helper streptomycete, Micromonospora ‐strain BCRU‐MM18 and grown for 12 weeks. The root nodules are easily visible by the naked eye. In the current issue this symbiotic interaction of gram‐positive bacteria and plant roots is described in more detail (Foto: Gernot Vobis, San Carlos de Bariloche, Argentina). (© 2007 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Infectivity variation ofDiscaria trinervis‐nodulatingFrankiain Patagonian soil according to season and storage conditions

Changes in the infectious capacity of soilborneFrankiafrom the same site may depend on environmental conditions. To test this, we examined the effect of season of sampling, sample storage protocol and storage time. The nodulation capacity ofFrankiafrom rhizospheric soils ofDiscaria trinervis(Hook et Arn.) Reiche (Rhamnaceae) growing in northwest Patagonia (Argentina) was measured using the most probable number method. Soil samples were collected seasonally and either stored moist at 4°C or air dried at room temperature for few days. Old (air‐dried) soil samples were also assayed. All soils nodulatedD.trinervisseedlings. Nodulation units (NU) ranged from 44 (spring, moist storage) to about 1 ml−1of soil (summer moist, and summer and autumn, air‐dried storage), with intermediate values in other samples. Soils stored for 12 years, 6 months or 1 week had similar NU.FrankiaNU positively correlated with soil water content (r= 0.6,P&lt; 0.05); therefore, it is likely that soil moisture is a relevant factor regulating soilborneFrankianodulation ability in Patagonian soils. We suggest thatFrankiacan remain as spores or grow saprophytically in Patagonian soils.