Host range ofFrankia endophytes

Summary Cross-inoculation experiments with 10 pure cultured strains and 17 host species were carried out. The 10 strains were isolated from the root nodules on actinorhizal trees ranging in 9 species, 5 genera and 4 families. The host species belong to 5 genera. The pure cultured strains fromAlnus are of strong ability to infect different species of the same genus. The seedlings inoculated with these strains are able to nodulate normally. These strains can also infect and nodulate the seedlings ofMyrica californica, but not the seedlings of Elaeagnus, Casuarina andMyrica rubra. The pure cultured strains from Elaeagnus can infect and nodulate the host species in the same genus and family with an exception ofE. viridis vardelavayi, which can be only poorly nodulated by a few strains from Elaeagnus. The strains from Elaeagnus cannot infect the seedlings of Alnus andMyrica rubra. The results presented here suggest thatFrankia endophytes can be divided into two groups: Alnus group and Elaeagnus group.     

Nodulation patterns of actinorhizal plants in the family rosaceae

Patterns of nodulation, growth, andFrankia — host specificity have not been well characterized for the actinorhizal genera in the family Rosaceae because of the scarcity ofFrankia isolates from these taxa. Furthermore, the few isolates available from actinorhizal Rosaceae have consistently failed to nodulate plants from the host genus. In a series of experiments, species of rosaceousDryas, Cowania, Cercocarpus, Fallugia, andPurshia were inoculated withFrankia isolates, crushedDryas actinorhizae, and neoglacial soils to ascertain whether any of these inocula would effectively induce nodulation. Neoglacial soils from Alaska and Canada nodulated not only the localDryas drummondii, but alsoCercocarpus betuloides, Cowania mexicana andPurshia tridentata from distant and ecologically diverse locales as well as nonrosaceous, actinorhizal species ofAlnus, Elaeagnus, Myrica, andShepherdia. But of eightFrankia isolates, including two fromPurshia tridentata and one fromCowania mexicana, none were able to induce nodulation onPurshia orCowania species. Globular, actinorhizae-like nodules incapable of acetylene reduction were produced onC. betuloides inoculated withFrankia isolates. Crushed nodule suspensions fromDryas drummondii nodulated rosaceousCowania, Dryas andPurshia, as well as non-rosaceousElaeagnus, Myrica, andShepherdia species. Nodules produced by inoculation ofCowania mexicana andPurshia tridentata with crushed, dried nodule suspensions fromDryas drummondii reduced acetylene to ethylene, indicating nitrogenase activity for these nodulated plants. These data suggest that a similar microsymbiont infects the actinorhizal genera in the family Rosaceae.     

Nodulation of actinorhizal plants byFrankia strains capable of both root hair infection and intercellular penetration

Summary A morphological analysis of the initiation and development of root nodules ofElaeagnus angustifolia andMyrica cerifera inoculated with pure-culturedFrankia strains DDB 011610 or DDB 020110 was undertaken. From ultrastructural observations it was determined that both of theseFrankia strains can infectElaeagnus by an intercellular penetration mechanism andMyrica by the root hair infection mechanism. This indicates that both of these strains have the ability to infect host plant roots by either of two mechanisms. The reverse, thatElaeagnus orMyrica could be infected by both mechanisms, was not observed. The infection and nodule development processes of these two plants in combination with these strains were similar to observations made in previous studies (Miller andBaker 1985,Torrey andCallaham 1979). However, one exception was identified in the development of the prenodule ofMyrica when infected with strain 011610, in that endophytic hyphae developed vesicles within the cells of the prenodule. This event has not been described before for any of the actinorhizal genera and may be an indication of less than optimal compatibility between the host plant and the symbiont.Contribution no. 876 of the Battelle-Kettering Laboratory.     

Large scale inoculation of actinorhizal plants withFrankia

Summary From 1979 to 1984 more than seven million seedlings of actinorhizal plants were successfully inoculated on an industrial scale withFrankia inoculants. Nodulated seedlings were produced in greenhouses to be used for land reclamation in northern Québec by the Societe d'Energie de la Baie James (SEBJ) and also by the City of Montréal for a revegetation program. Crushed-nodule homogenates andFrankia pure culture formulations were compared for large scale inoculation of green alder. Pure culture inoculant was found to be superior than crushed-nodule homogenates yielding reproducible nodulation of seedlings. Two inoculation methods of theFrankia pure culture inocula were compared: soil injection and spraying with greenhouse watering devices. Both methods resulted in efficient nodulation ofAlnus crispa, A. glutinosa, A. rugosa, Elaeagnus angustifolia, E. commutata, Hippophaë rhamnoides, Myrica gale andShepherdia argentea.     

The initiation,development and structure of root nodules inElaeagnus angustifolia L. (Elaeagnaceae)

Summary A correlated light and electron microscopic study was undertaken of the initiation and development of root nodules of the actinorhizal tree species,Elaeagnus angustifolia L. (Elaeagnaceae).Two pure culturedFrankia strains were used for inoculation of plants in either standing water culture or axenic tube cultures. Unlike the well known root hair infection of other actinorhizal genera such asAlnus orMyrica the mode of infection ofElaeagnus in all cases was by direct intercellular penetration of the epidermis and apoplastic colonization of the root cortex. Root hairs were not involved in this process and were not observed to be deformed or curled in the presence of the actinomyceteFrankia. In response to the invasion of the root, host cells secreted a darkly staining material into the intercellular spaces. The colonizingFrankia grew through this material probably by enzymatic digestion as suggested by clear dissolution zones around the hyphal strands. A nodule primordium was initiated from the root pericycle, well in advance of the colonizingFrankia. No random division of root cortical cells, indicative of prenodule formation was observed inElaeagnus. As the nodule primordium grew in size it was surrounded by tanninised cells of a protoperiderm. The endophyte easily traversed this protoperiderm, and once inside the nodule primordium cortex ramified within the intercellular spaces at multiple cell junctions. Invasion of the nodule cortical cells occurred when a hyphal branch of the endophyte was initiated and grew through the plant cell wall, again by apparent enzymatic digestion. The plant cell plasmalemma of invaded cells always remained intact and numerous secretory vesicles fused with it to encapsulate the advancingFrankia within a fibrous cell wall-like material. Once within the host cell some endophyte cells began to differentiate into characteristic vesicles which are the presumed site of nitrogen fixation. This study clearly demonstrates that alternative developmental pathways exist for the development of actinorhizal nitrogen-fixing root symbioses.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Occurrence of Myrica-nodulating Frankia in Hawaiian volcanic soils

The ability of Hawaiian volcanic soils to nodulate actinorhizal Myrica cerifera, Casuarina equisetifolia, and Alnus glutinosa was determined using a host-plant bioassay. Myrica-nodulating Frankia occurred in five volcanic deposits with depositional ages ranging from 20 to 162 years before present. The oldest deposit had a mean estimated nodulation capacity from 450 to 1200 times greater than those of the younger deposits. Only the oldest deposit had high moisture content, high organic matter content, and increased vegetative cover, including an abundance of actinorhizal M. faya. Casuarina- and Alnus-nodulating Frankia were not detected in any of these volcanic deposits.     

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

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.     

Nitrogenase activity in root nodule homogenates ofAlnus incana

Summary Root nodule homogenates of actinorhizal plants may representFrankia in a symbiotic stage but released from environmental influence of the host plant. Anaerobic homogenization with a blender in buffer supplied with sucrose, polyvinylpyrrolidone and reducing substances gave three times higher yields of nitrogenase activity (C₂H₂-reduction) than crushing the nodules in liquid nitrogen. The activity in the homogenates was very reproducible and was, on average, nearly twice as high as the activity in excised nodules and c. 10% of the activity in intact plants. The difference in activity between excised nodules and intact plants was, roughly by halves, due to removal of the root system from the pot and to excision of the nodules. The nitrogenase activity in the homogenates was slightly higher when nodule excision was done in Ar or under water as well as after treatment of the homogenate with toluene or Triton X-100 or osmotic shock. These gains in activity were considered too small to outweigh the increased complications of preparing homogenates for routine use. Due to the reproducible recovery of nitrogenase in the homogenates the technique seems useful for physiological studies on nitrogen fixation inAlnus incana.     

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

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 comparison of symbiotic nitrogen fixation in Scotland inAlnus glutinosa andAlnus rubra

Summary Alnus glutinosa andAlnus rubra growing in the field in Scotland show specific nitrogenase activities of the same order of magnitude. The period of maximum potential nitrogenase activity coincides with that of maximum growth in late Spring and Summer. It is suggested that the retention of nitrogenase activity into the Autumn when growth has virtually ceased may be important as a contribution to the nitrogenous reserves of the tree.Bioassay of different Scottish soils, all collected from the locality of natural stands ofAlnus glutinosa, showed wide variation in the nodulation of seedlings, although generally a soil poor for nodulation ofAlnus glutinosa generally gave poor nodulation ofAlnus rubra. Soils of pH 4.5 to 6.5, best suited for growth and nitrogen fixation of the two species, often gave nodules showing highest specific nitrogen fixing activity. Young (2 to 3 year old) plants in glasshouse or controlled environment cabinet, inoculated withAlnus glutinosa endophyte, differed from mature field grown plants, however, sinceAlnus rubra required a much larger (up to 2.5 times) mass of root nodules to fix a unit quantity of N. Microscopic comparison of the nodules of glasshouse plants showed that the proportion of cells containing the vesicular (nitrogen fixing) form of the endophyte was only slightly lower inAlnus rubra than inAlnus glutinosa and it is suggested that the differences in specific nitrogen fixing activity between the two species may reflect some incompatibility of function of theAlnus glutinosa endophyte when in symbiosis withAlnus rubra.     

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

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

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. This revised version was published online in June 2006 with corrections to the Cover Date.     

Soil properties and actinorhizal vegetation influence nodulation of Alnus glutinosa and Elaeagnus angustifolia by Frankia

Nodulation (mean number of nodules per seedling) was 5 times greater for Elaeagnus angustifolia than for Alnus glutinosa overall when seedlings were grown in pots containing either an upland or an alluvial soil from central Illinois, USA. However, the upland Alfisol had 1.3 times greater nodulation capacity for A. glutinosa than for E. angustifolia. The presence of A. glutinosa trees on either soil was associated with a two-fold increase in nodulation capacity for E. angustifolia. Nodulation increases for soils under A. glutinosa were obtained for A. glutinosa seedlings in the Alfisol, but decreased nodulation for A. glutinosa seedlings occurred in the Mollisol. Greatest nodulation of E. angustifolia seedlings occurred near pH 6.6 for soil pH values ranging from 4.9 to 7.1, while greatest nodulation of A. glutinosa occurred at pH 4.9 over the same pH range. Nodulation was not affected by total soil nitrogen concentrations ranging from 0.09 to 0.20%. Mollisol pH was significantly lower under A. glutinosa trees than under E. angustifolia trees. For 4- to 8-year-old field-grown trees, A. glutinosa nodule weights were negatively correlated with soil pH, while for similar aged E. angustifolia trees nodulation in the acidic Alfisol was not detected.     

Nitrogen fixation byAlnus viridis (Chaix) DC.

Summary InAlnus viridis nodule growth relative to plant growth was inversely related to the quantity of nitrate added to nutrient solutions. Nodulated plants showed maximum growth when grown independently of supplied nitrogen and made better growth in its absence than unnodulated plants at any level of added nitrogen. Low levels of nitrate caused a depression of growth of nodulated plants, apparently by suppressing both nitrogen fixation and nodule growth. Nodules in nitrogen-free sand culture fixed atmospheric nitrogen at a rate of 6.6 mg/day/g nodule. Phosphorus deficiency was induced by low levels of phosphate and resulted in small plants with dark-green foliage. Root and nodule growth as a percentage of total plant growth and the percentage of total accumulated plant nitrogen below ground were greater at a root temperature of 11°C than 21°C. Thus at low root temperature processes other than nitrogen fixation were limiting to plant growth. Excised nodules were exposed to an N ₂ ¹⁵ -enriched atmosphere. A positive correlation between rate of nitrogen fixation and temperature was obtained, with optimum fixation occurring at about 20°C. It was shown that in spite of decreasing mean temperatures with increase in altitude, rate of nitrogen fixation by nodules of plants growing in the field increased with increase in altitude. This latter trend was deduced to be a reflection of the extent to which the field sites were nitrogen deficient in relation to climatically possible growth.     

Sucrose synthase and enolase expression in actinorhizal nodules ofAlnus glutinosa: comparison with legume nodules

Two different types of nitrogen-fixing root nodules are known — actinorhizal nodules induced byFrankia and legume nodules induced by rhizobia. While legume nodules show a stem-like structure with peripheral vascular bundles, actinorhizal nodule lobes resemble modified lateral roots with a central vascular bundle. To compare carbon metabolism in legume and actinorhizal nodules, sucrose synthase and enolase cDNA clones were isolated from a cDNA library, obtained from actinorhizal nodules ofAlnus glutinosa. The expression of the corresponding genes was markedly enhanced in nodules compared to roots. In situ hybridization showed that, in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the central vascular bundle of a nodule lobe. Legume sucrose synthase expression was studied in indeterminate nodules from pea and determinate nodules fromPhaseolus vulgaris by usingin situ hybridization.     

Inoculation and production of container-grown red alder seedlings

Summary The inoculation ofAlnus rubra (red alder) withFrankia sp. can lead to a highly efficient symbiosis. Several factors contribute to the successful establishment of nitrogenfixing nodules: (1) quantity and quality ofFrankia inoculant; (2) time and method of inoculation; (3) nutritional status of the host plant.Frankia isolates were screened for their ability to nodulate and promote plant growth of container-grown red alder. Inoculations were performed on seedlings and seeds. Apparent differences in symbiotic performance could be seen when seeds or seedlings were inoculated. Plants inoculated at planting performed significantly better than those inoculated four weeks later in terms of shoot height, nodule number and shoot dry weight. If inoculation was delayed further, reduction in shoot height, nodule number and shoot dry weight resulted. The effect of fertilizer was also investigated with regard to providing optimal plant growth after inoculation. Plants receiving 1/5 Hoagland's solution minus nitrogen showed maximal plant growth with abundant nodulation. Plants receiving 1/5 Hoagland's solution with nitrogen showed excellent plant growth with significantly reduced nodulation.     

PCR-restriction fragment length polymorphism identification and host range of single-spore isolates of the flexible Frankia sp. strain UFI 132715.

Twelve single-spore isolates of the flexible Elaeagnus-Frankia strain UFI 132715 fulfilled the third and the fourth of Koch's postulates on both Alnus and Elaeagnus axenic plants. Seminested nifD-nifK PCR-restriction fragment length polymorphisms provided evidence for the genetic uniformity of the single-spore frankiae with the mother strain and its plant reisolates and allowed their molecular identification directly inside Alnus and Elaeagnus nodules. The clonal nature of these single-spore-purified frankiae should allow safe mutagenesis programs, while their flexible phenotype makes them a powerful tool for understanding the molecular interactions between Frankia strains and actinorhizal plants and for identifying Frankia nodulation genes.     

Effects of continuous nitrogen application and nitrogen preconditioning on nodulation and growth ofCeanothus griseus varhorizontalis

Rooted cuttings ofCeanothus griseus varhorizontalis were irrigated with 0, 10, 20, 50, 75 or 100ppm nitrogen as NH₄NO₃ for eight weeks prior to inoculation with infectiveFrankia. After inoculation, half of the plants for each treatment nitrogen level continued to be irrigated with the preconditioning nitrogen level and half were given no more supplemental nitrogen. For plants continuously receiving nitrogen, nodule initiation (nodule number) was inversely correlated with increasing supplemental nitrogen levels, and suppressed above 50 ppm N. Leaf nitrogen above 2% in continuous-N plants correlated with greatly reduced or suppressed nodulation. Plants maintained after inoculation without supplemental nitrogen showed influence of the prior nitrogen treatment on nodulation. Preconditioning at 50 ppm and above greatly reduced the number of nodules formed. The evidence suggests that stored internal nitrogen can regulate nodulation.Plant biomass accumulated maximally when nodulation was suppressed, at 75 and 100 ppm supplemental N applied continuously. Internode elongation during the nodulation period occurred only on nodulated plants, or in the presence of supplemental N (10 ppm and above).     

Influence of combined nitrogen level andEucalyptus competition on dinitrogen fixation in nodulatedCasuarina

Summary Experiments were conducted to determine the effect on biological dinitrogen fixation byCasuarina of available nitrogen (N) in the substrate and competition by interplantedEucalyptus. In these experiments, combined N was applied to the plants after nodules were developed and functioning. Both environmental factors, nitrate and competition, were observed to influence biological dinitrogen fixation byCasuarina, but not yield (total dry weight). In one experiment, the proportion of nitrogen derived byCasuarina from atmospheric fixation (pNdfa) was observed to be inhibited by potassium nitrate in a linear fashion. However, substrate N did not significantly affect the weight of root nodules. Thus nodule dry weight was not highly correlated with the proportion of nitrogen fixed. In a second experiment, the presence of a non-fixing interplanted species,Eucalyptus, increased dinitrogen fixation inCasuarina.Casuarina interplanted withEucalyptus obtained a greater proportion of its nitrogen (94.75%) from fixation than didCasuarina grown alone (86.68%) suggesting that competition for substrate N influences the proportion of nitrogen fixed by this actinorhizal plant.Dedicated to the memory of Professor John G. Torrey