Actinorhizal Plant Species Research Articles

Chitotetraose activates the fungal-dependent endosymbiotic signaling pathway in actinorhizal plant species.

Mutualistic plant-microbe associations are widespread in natural ecosystems and have made major contributions throughout the evolutionary history of terrestrial plants. Amongst the most remarkable of these are the so-called root endosymbioses, resulting from the intracellular colonization of host tissues by either arbuscular mycorrhizal (AM) fungi or nitrogen-fixing bacteria that both provide key nutrients to the host in exchange for energy-rich photosynthates. Actinorhizal host plants, members of the Eurosid 1 clade, are able to associate with both AM fungi and nitrogen-fixing actinomycetes known as Frankia. Currently, little is known about the molecular signaling that allows these plants to recognize their fungal and bacterial partners. In this article, we describe the use of an in vivo Ca2+ reporter to identify symbiotic signaling responses to AM fungi in roots of both Casuarina glauca and Discaria trinervis, actinorhizal species with contrasting modes of Frankia colonization. This approach has revealed that, for both actinorhizal hosts, the short-chain chitin oligomer chitotetraose is able to mimic AM fungal exudates in activating the conserved symbiosis signaling pathway (CSSP) in epidermal root cells targeted by AM fungi. These results mirror findings in other AM host plants including legumes and the monocot rice. In addition, we show that chitotetraose is a more efficient elicitor of CSSP activation compared to AM fungal lipo-chitooligosaccharides. These findings reinforce the likely role of short-chain chitin oligomers during the initial stages of the AM association, and are discussed in relation to both our current knowledge about molecular signaling during Frankia recognition as well as the different microsymbiont root colonization mechanisms employed by actinorhizal hosts.

Nitrogen fixation by riparian plants belonging to Coriariaceae, Rhamnaceae, and Gunneraceae in Northwest Patagonia

Nitrogen fixation by symbiotic bacteria associated with different plant species is a key process of natural ecosystems. To better understand the role of native N2-fixing species in the N economy of riparian ecosystems in northwest Patagonia (Argentina), we evaluated: 1) foliar δ15N and N concentrations of actinorhizal Coriaria ruscifolia, Discaria chacaye, and Colletia hystrix, several non-actinorhizal plants (including Gunnera chilensis), and associated soils in riparian forest sites; 2) the proportion of N derived from the atmosphere (Ndfa) of the actinorhizal plant species from riparian forest and of D. chacaye and Ochetophila trinervis from steppe sites; and 3) trends of foliar and soil δ15N and N concentration with mean annual precipitation (MAP). Although soil N concentrations and δ15N did not vary among plant species within any of the sites, foliar N concentration and δ15N differed among species. In general, N2-fixing species had higher foliar N concentrations and δ15N values closer to 0, the atmospheric value. Both variables separated the groups of N2-fixing and non-N2-fixing plant species. Foliar and soil δ15N correlated positively for non-N2-fixing species but not for N2-fixing species. Across all sites, the Ndfa of C. ruscifolia and C. hystrix was ~100%, ~75% for D. chacaye, and ~50% for G. chilensis. For all species, foliar N concentration and soil δ15N was negatively correlated with MAP, but only non-N2-fixing species showed a significant correlation of foliar δ15N with MAP. These data suggest that plant available N decreases as MAP increases but with no effect on N2-fixing species.

Frankia communities at revegetating sites in Mt. Ontake, Japan.

In 1984 at Mt. Ontake in Japan, an earthquake caused a devastating landslide, and as a result, the vegetation on the south slope of the mountain was completely eliminated. In higher elevation (2000m) areas, revegetation has not yet been completed even 30years after the landslide. Revegetation progress throughout the area was heterogeneous. In the partially revegetated areas, actinorhizal plant species such as Alnus maximowiczii and Alnus matsumurae have been found. In the present study, we investigated the Frankia communities in the higher-elevation area using sequence analysis of the amplified nifH (dinitrogenase reductase) gene from nodule and soil samples collected in the disturbed region, undisturbed forest, and in the boundary between the disturbed region and the undisturbed forest. Phylogenetic analysis of partial nifH sequences revealed the presence of six clusters, each of which consisted of highly similar (> 99%) sequences. Four clusters showed significant sequence similarity to Frankia (three Alnus- and a Casuarina-infecting strains). Diversity in the Frankia community was relatively low-only one or two clusters were detected in a site. At most of the sampling sites, a dominant cluster in a nodule coincided with that in rhizosphere soil, indicating that community structure in the rhizosphere is a primary factor that determines occupancy in a nodule. No significant difference in community structure was observed between plant species. Diversity in the Frankia community varied depending on revegetation progress. Cluster A, which was the most dominant in the disturbed region, was likely to have invaded from undisturbed forest.

Effect of Pinus ponderosa afforestation on soilborne Frankia 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.

Recent advances in actinorhizal symbiosis signaling.

Nitrogen and phosphorus availability are frequent limiting factors in plant growth and development. Certain bacteria and fungi form root endosymbiotic relationships with plants enabling them to exploit atmospheric nitrogen and soil phosphorus. The relationships between bacteria and plants include nitrogen-fixing Gram-negative proteobacteria called rhizobia that are able to interact with most leguminous plants (Fabaceae) but also with the non-legume Parasponia (Cannabaceae), and actinobacteria Frankia, which are able to interact with about 260 species collectively called actinorhizal plants. Fungi involved in the relationship with plants include Glomeromycota that form an arbuscular mycorrhizal (AM) association intracellularly within the roots of more than 80% of land plants. Increasing numbers of reports suggest that the rhizobial association with legumes has recycled part of the ancestral program used by most plants to interact with AM fungi. This review focuses on the most recent progress made in plant genetic control of root nodulation that occurs in non-legume actinorhizal plant species.

Validation of candidate reference genes for qRT-PCR studies in symbiotic and non-symbiotic Casuarina glauca Sieb. ex Spreng. under salinity conditions

Casuarina glauca is a model actinorhizal plant species that establishes N2-fixing symbiosis with Frankia bacteria. This plant is highly resilient to extreme environments, being commonly found in saline zones. Gene expression studies by quantitative real-time polymerase chain reaction (qRT-PCR) constitute a powerful tool to analyze the mechanisms underlying plant stress-tolerance. One of the crucial steps of this technique is the selection and validation of reference genes to produce accurate data. In this work we report on the evaluation of a set of ten reference genes to be used in qRT-PCR studies in C. glauca grown under high salt concentrations, following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Five independent methods (geNorm, NormFinder, BestKeeper, Coefficient of Variance, and ReFinder) were used to evaluate gene stability. According to the results, the calibration of qRT-PCR reactions with the most versus the least stable reference genes produced different expression patterns of C. glauca stress responsive genes (CgCS and CgAPX). The same was observed when data was normalized with one, two or three stable reference genes. These study constitutes a baseline for accurate qRT-PCR analysis in C. glauca exposed to high salt concentrations which should include the use of at least two stable reference genes.

Indigenous actinorhizal plants of Australia

Indigenous species of actinorhizal plants of Casuarinaceae, Elaeagnaceae and Rhamnaceae are found in specific regions of Australia. Most of these plants belong to Casuarinaceae, the dominant actinorhizal family in Australia. Many of them have significant environmental and economical value. The other two families with their indigenous actinorhizal plants have only a minor presence in Australia. Most Australian actinorhizal plants have their native range only in Australia, whereas two of these plants are also found indigenously elsewhere. The nitrogen-fixing ability of these plants varies between species. This ability needs to be investigated in some of these plants. Casuarinas form a distinctive but declining part of the Australian landscape. Their potential has rarely been applied in forestry in Australia despite their well-known uses, which are being judiciously exploited elsewhere. To remedy this oversight, a programme has been proposed for increasing and improving casuarinas that would aid in greening more regions of Australia, increasing the soil fertility and the area of wild life habitat (including endangered species). Whether these improved clones would be productive with local strains of Frankia or they need an external inoculum of Frankia should be determined and the influence of mycorrhizal fungi on these clones also should be investigated.

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.

An overview of actinorhizal plants in Africa.

A compilation and synthesis of information derived from plant databases and other sources on the occurrence, diversity and geographic distribution of actinorhizal plants in Africa is presented in this review. Actinorhizal plants are a specific group of non-leguminous, woody dicots having symbiotic, nitrogen-fixing root nodules that are induced on roots of actinorhizal plant species by soil actinomycetes of the genus Frankia. There is a lack of basic information on actinorhizal plants in Africa compared with other major land masses in the world. Results indicate that most, if not all, African countries and climatic regions have native or introduced actinorhizal species. A synthesis of available information indicates that there are six families, nine genera and 38 reported species of actinorhizal plants in Africa. Of these, 21 species are native and 17 are exotic. The families and corresponding number of species in each genus are: Betulaceae (native Alnus glutinosa (1), exotic Alnus (2)); Casuarinaceae (exotic Casuarina (5), exotic Allocasuarina (3), exotic Gymnostoma deplancheana (1)); Coriariaceae (native Coriaria myrtifolia (1)); Myricaceae (native Morella (19), exotic Morella cerifera (1)); Rhamnaceae (exotic Ceanothus caeruleus (1), exotic Colletia paradoxa (1)); and Elaeagnaceae (exotic Eleaegnus angustifolia (1)). Four reports of native, actinorhizal Ceanothus species in Africa found in the database were determined to be false, instead, being non-actinorhizal species. Widespread plantings of exotic Casuarinaceae have been introduced into tropical and arid zones of Africa as multipurpose trees, especially in arid regions where native species do not occur. There is a diverse assemblage of native species of Morella in Africa, mostly shrubs or small trees, which provide medicine, other useful chemicals and wildlife habitat. Many native Morella species are isolated in montane islands, apparently leading to greater speciation than in Eurasia from where the genus migrated into Africa. The current status and knowledge of African actinorhizal plants indicates a need to focus research on their biogeography, biology, ecology, genetics and use.

Interactions among soil biology, nutrition, and performance of actinorhizal plant species in the H.J. Andrews Experimental Forest of Oregon

The study examined the effect of Frankia, macronutrients, micronutrients, mycorrhizal fungi, and plant-growth-promoting fluorescent Pseudomonas sp. on total biomass, nodule weight, and nitrogen fixation of red alder ( Alnus rubra) and snowbrush ( Ceanothus velutinus) under greenhouse conditions. The soil samples were collected from a 10-year-old clearcut on the H.J. Andrews Experimental Forest, Oregon. Within the clearcut, four sampling points were selected along a slope gradient. Red alder and snowbrush plants were greenhouse-grown in a mix of soil–vermiculite–perlite (2:1:1) for 6 and 12 months, respectively. Plants were inoculated with Frankia and a fluorescent Pseudomonas sp. Some of the red alder were also inoculated with ectomycorrhizal Alpova diplophloeus, and some snowbrush with endomycorrhizal Glomus intraradix. There was no interaction between treatment and slope location for either species. There were significant treatment effects for red alder, but not for snowbrush. Red alder seedlings given Frankia and macronutrients produced more biomass and had greater nitrogen fixation than seedlings grown without additions; adding A. diplophloeus increased nitrogen fixation by 33% over that obtained with Frankia plus macronutrients. Frankia, macronutrients, and the mycorrhizal fungus together increased nitrogen fixation by 136% over the control. Adding only micronutrients to Frankia and macronutrients, however, reduced nitrogen fixation by nearly one half; the presence of the mycorrhizal fungus appeared to buffer these negative effects. Pseudomonas inoculation did not affect any of the measured variables. Slope location of soil affected the two plant species differently. Red alder seedlings grown in upper slope soil had greater biomass and nitrogen fixation than those grown in soil from the lower slope. In contrast, snowbrush plants had greater biomass, nodule weight, and nitrogen fixation when grown in bottom slope soil rather than on soil from any of the other slope positions.

Effect of elevated carbon dioxide in the root atmosphere on nitrogenase activity in three actinorhizal plant species

In wet soils, nitrogen-fixing root nodules are subjected to elevated CO2. Only a few studies have examined the effect of elevated CO2 on nitrogenase activity, and the results have been mixed. We examined intact black alder (Alnus glutinosa (L.) Gaertn.) and sweet gale (Myrica gale L.) seedlings and field-collected speckled alder (Alnus incana ssp. rugosa (Du Roi) Claus.) nodules to clarify the effects of elevated CO2. Nitrogenase activity was measured via acetylene reduction in an open, flow-through system. We found that repeated measurements of the peak rate of nitrogenase activity, the only reliable measure of nitrogenase activity, could be made on the same plant via 150-s exposures to acetylene separated by 20 min without acetylene. Our results for elevated CO2 consistently showed that it had little effect on nitrogenase activity at low concentrations and increasingly inhibited nitrogenase activity as the CO2 concentration increased. In black alder, 0.5 kPa CO2 had little effect, whereas 3.0 kPa CO2 reduced nitrogenase activity 31–35%. Sweet gale, was less sensitive to elevated CO2 and was unaffected by 1.5 kPa CO2. Black alder grown with the roots, but not the shoots, in 1.3 kPa CO2 showed only minimal acclimation to elevated CO2.Key words: acetylene reduction technique, actinorhizal plants, Alnus, carbon dioxide, Myrica gale, nitrogen fixation.

Diversity and specificity of Frankia strains in nodules of sympatric Myrica gale, Alnus incana, and Shepherdia canadensis determined by rrs gene polymorphism.

The identity of Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct from Frankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from two M. gale nodules at a second site in Wisconsin. Frankia strains from nodules of S. canadensis belonged to a divergent subset of a cluster of Elaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankia populations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankia populations found in Myrica nodules.

Effect of elevated carbon dioxide in the root atmosphere on nitrogenase activity in three actinorhizal plant species

Effect of elevated carbon dioxide in the root atmosphere on nitrogenase activity in three actinorhizal plant species

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

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

Transient Responses of Nitrogenase to Acetylene and Oxygen in Actinorhizal Nodules and Cultured Frankia

Nitrogenase activity in root nodules of four species of actinorhizal plants showed varying declines in response to exposure to acetylene (10% v/v). Gymnostoma papuanum (S. Moore) L. Johnson. and Casuarina equisetifolia L. nodules showed a small decline (5-15%) with little or no recovery over 15 minutes. Myrica gale L. nodules showed a sharp decline followed by a rapid return to peak activity. Alnus incana ssp. rugosa (Du Roi) Clausen. nodules usually showed varying degrees of decline followed by a slower return to peak or near-peak activity. We call these effects acetylene-induced transients. Rapid increases in oxygen tension also caused dramatic transient decreases in nitrogenase activity in all species. The magnitude of the transient decrease was related to the size of the O(2) partial pressure (pO(2)) rise, to the proximity of the starting and ending oxygen tensions to the pO(2) optimum, and to the time for which the plant was exposed to the lower pO(2). Oxygen-induced transients, induced both by step jumps in pO(2) and by O(2) pulses, were also observed in cultures of Frankia. The effects seen in nodules are purely a response by the bacterium and not a nodule effect per se. Oxygen-induced nitrogenase transients in actinorhizal nodules from the plant genera tested here do not appear to be a result of changes in nodule diffusion resistance.

Effects of substrate nitrate concentration on symbiotic nodule formation in actinorhizal plants

The response ofAlnus glutinosa, Casuarina cunninghamiana, Elaeagnus angustifolia andMyrica cerifera to a range of substrate nitrogen levels in solution, in relation to plant growth, infection, nodulation and root fine structure was studied. Nine concentrations of potassium nitrate ranging from 0.05 to 3.0 mM, were tested on each of the species. Plants were inoculated withFrankia pure cultures after a two week exposure to one of the nine levels of added nitrate. After six more weeks with constant exposure to nitrate, plants were harvested and assayed. With the exception of Myrica, regression analyses of whole plant dry weights as a function of added nitrate were highly significant. There was a tendency for nodulated plants grown at intermediate levels of added nitrate to exhibit higher relative growth rates, probably due to the additive effect of substrate nitrogen and fixation of atmospheric nitrogen. The mean numbers of nodules per plant were, with the exception of Alnus, significantly higher at intermediate levels of added nitrate, as were mean nodule dry weights. A highly significant inverse relationship between nodule weight as a percentage of whole plant weight was found in Elaeagnus and Myrica. The observed response of Elaeagnus to added nitrate compared to other actinorhizal plants appears to demonstrate that root hair infected plants are much more sensitive to the inhibitory effects of added nitrate than plants infected by intercellular penetration. A sharp reduction in the presence of root hairs at high concentrations of nitrate was observed. This indicates that the inhibition of nodulation in some actinorhizal plant species results from nitrate induced root hair suppression.

Relationships among pure cultured strains of Frankia based on host specificity

Fifty strains of Frankia were tested for their ability to nodulate six species of actinorhizal plants. Pure cultured strains were used to inoculate seedlings of Alnus glutinosa (L.) Gaertn., Alnus rubra Bong., Casuarina equisetifolia L., Elaeagnus angustifolia L., Hippophaë rhamnoides L. and Myrica cerifera L. in nutrient solution culture. From the results of this study, host inoculation groups among the actinorhizal plants were defined. Although overlap between host inoculation groups appears to be common, the results from this study did not support the view that Frankia strains are promiscuous. All Frankia strains tested in this study could easily be classified into four major host‐specificity groups.

Leghemoglobin-like sequences in the DNA of four actinorhizal plants

A cloned cDNA partial copy of a soybean leghemoglobin mRNA was used to probe genomic DNA of four species of actinorhizal plants. Southern blot hybridization revealed the presence of sequences with homology to the leghemoglobin probe in DNA from Alnus glutinosa, Casuarina glauca, Ceanothus americanus and Elaeagnus pungens. The hybridization patterns of the restriction fragments revealed some fragment size conservation between the DNA of soybean and the DNA of four actinorhizal plants which are taxonomically unrelated to soybean or to each other. The results presented here indicate that globin gene sequences are much more widely distributed in the plant kingdom than has previously been thought. Furthermore, if sequence conservation is actually as high as the restriction fragment patterns suggest, the evolution of the DNA surrounding the globin sequences has been highly constrained.

Cataloging Frankia strains

A catalog listing known Frankia isolates and introducing a homogeneous numbering system for all strains will be published and distributed to interested scientists. Details are given concerning the procedure to be followed for cataloging the strains as well as the coding of information about them. An up-to-date listing of actinorhizal plant species is included along with the codes to be used for each plant taxon.