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.     

Heterologous hybridization of Frankia DNA to Rhizobium meliloti and Klebsiella pneumoniae nif genes

A nif gene probe from Rhizobium meliloti was used to isolate a recombinant bacteriophage from a Frankia sp. ArI3 gene bank. There is a large homology between nif D and nif H genes of R. meliloti or Klebsiella pneumoniae and Frankia DNA sequences. Approximately 4.5 kb to the right of nif K, we have localized a DNA region hybridizing to a R. meliloti probe containing nif A and nif B genes. The extent of the homology was greater for nif B than for nif A.     

Occurrence and activity of hydrogenase in symbiotic Frankia from field-collected Alnus incana

Occurrence and activity of the hydrogen uptake enzyme were studied in root nodule homogenates made from plants of Alnus incana (L.) Moench collected from field sites in the northern part of Sweden. Nitrogenase (EC 1.7.99.2) activity (estimated by acetylene reduction) and hydrogen evolution were studied in excised nodules. All Frankia sources showed acetylene reduction activity, and possessed a hydrogen uptake system. Hydrogen uptake in nodule homogenates from the Frankia sources measured at 23.8 μM H₂ ranged from 0.04 to 5.0 μmol H₂ (g fresh weight nodule)⁻¹ h⁻¹. The H₂ uptake capacity of nodule homogenates from one of the Frankia sources was almost 8 times higher than the hydrogen evolution from nitrogenase, both expressed on a nodule fresh weight basis. Frankia sources from field sites 6 and 11 showed K_m for H₂ of 13.0 and 23.6 μM H₂, respectively. This indicates similarities in the hydrogen uptake enzymes in the two Frankia sources. It is concluded that hydrogen uptake is a common characteristic in Frankia.     

Flavonoid-like compounds from seeds of red alder (Alnus rubra) influence host nodulation by Frankia (Actinomycetales)

Nitrogen-fixing root nodules are formed by Frankia spp. (Actinomycetales) on dicotyledonous hosts such as alders ( Alnus spp.). Flavonoid-containing preparations from seed washes of red alder ( Alnus rubra Bong.), and individual compounds isolated from such preparations, influenced nodulation of A. rubra by Frankia. Nodulation was enhanced by one flavonoid-like compound, and apparently inhibited by two other such compounds. Four flavonoid-like compounds had no significant effect on nodulation. The seven individual compounds purified from the seed washes were characterized spectrally as possible flavanones and isoflavones. Both the enhancer and the inhibitors appeared to be possible flavanones.     

The effectivity of Frankia for nodulation and nitrogen fixation in Alnus rubra and A. glutinosa

The effectivity of nodulation of Alnus rubra Bong, by Frankia isolates from A. rubra and Alnus glutinosa (L.) Gaertn. in Northern Britain was compared with strains from The Netherlands and North America, using plants grown in combined nitrogen-free conditions. All strains gave rise to spore (-) nodules, even when isolated from nodules from sites known to contain spore (+) nodules. Nodules of all plants evolved little hydrogen, probably due to the presence of an efficient uptake hydrogenase in the microsymbkmts. Nodule weight as a percentage of whole plant weight was higher for nodules of low specific activity (N fixed per unit weight nodules), attaining a maximum of 5.1% of plant dry weight in the least effective of the heterologous associations of A. glutinosa Frankia with A. rubra . The range of variation in nodule specific activity was much greater in heterologous than homologous associations, but nodules of high specific activity were found in both associations. However, plants that fixed most N during the growth period were not those with nodules of highest specific activity. The most effective associations were homologous symbioses, which combined good nodule growth per plant with satisfactory specific activity, fixing N at rates which would support superior plant growth under the prevailing growth conditions. Preliminary field experiments suggest that the most effective of the A. rubra isolates is suitable for use as an inoculant in nurseries. Strains isolated from A. glutinosa were more effective and showed a different order of effectivity in homologous symbioses compared with their association with A. rubra . An A. glutinosa strain was isolated, which stimulated satisfactory nodule growth and gave good nodule specific activity in both A. rubra and A. gtutinosa .     

Phylogenetic relationships among actinorhizal plants. The impact of molecular systematics and implications for the evolution of actinorhizal symbioses

A review of recent molecular systematic studies of actinorhizal plants and their Frankia endosymbionts is presented. For comparative purposes, a discussion of recent studies pertaining to the evolution of nodulation in the legume-rhizobium system is included. Molecular systematic studies have revealed that actinorhizal plants are more closely related than current taxonomic schemes imply. Broad-based analyses of the chloroplast gene rbcL indicate that all symbiotic root-nodulating higher plants belong to a single large clade. More focused molecular analyses of both legume and actinorhizal hosts within this large clade indicate that symbioses have probably arisen more than once. By comparing host phylogenies and recently published bacterial phylogenies, we consider the coevolution of bacterial symbionts with their actinorhizal hosts.     

Endophyte sporulation in root nodules of actinorhizal plants

All strains of isolated Frankia possess the genetic capacity to form sporangia since, when grown in vitro, they usually sporulate freely, depending on the physical and chemical environment in which they are cultured. Endophytic sporulation involving Frankia differentiation of sporangia within root nodules has been described in only 16 host species in 9 genera within six families of actinorhizal plants. From studies published to date, endophytic sporulation cannot be correlated with specific environmental conditions surrounding the host plants. Based on the literature and on previously unpublished observations from field and greenhouse studies, an account is given of the occurrence of sporulation in actinorhizal plants with emphasis on Alnus, Casuarina, Comptonia, Elaeagnus and Myrica . The possible role of the host plant in controlling Frankia sporulation as contrasted to the control exerted by the genetic constitution of the microbial symbiont is explored.     

Frankia菌在非豆科放线结瘤植物根际内的存活与分布

提取Ｆｒａｎｋｉａ菌１６ＳｒＲＮＡ,制备具专一性的寡核苷酸探针,通过同源杂交,在人工接种的木麻黄根际内检测出痕量Ｆｒａｎｋｉｓ菌结果表明,Ｆｒａｎｋｉａ可在环境因子的作用下被动迁移不论接种点的位置如何,经６个月的时间稳定后,其分布状态基本相同,主要集中于土层下８～３０ｃｍ处     

The use of PCR-based typing methods to assess the diversity of Frankia nodule endophytes of the actinorhizal shrub Ceanothus

Our understanding of the actinorhizal symbiosis, in particular of the Frankia-Ceanothus association, has been hampered by the failure to isolate infective strains in pure culture. Recently, the polymerase chain reaction (PCR) has been utilized to amplify regions of the Frankia genome, allowing analysis of the microsymbiont genome without first isolating the microbe in pure culture. Root nodules were collected from six Ceanothus spp. common to the coastal regions of the Santa Monica Mountains of southern California. Individual lobes were surface-sterilized, total DNA was extracted and amplified using prokaryotic-specific primers. To assess the genetic diversity of Frankia endophytes in the population studied, the BOX primer was used to generate genomic fingerprints of prokaryotic nodule inhabitants using rep-PCR. Fingerprint patterns fell into twelve distinct groups indicating the occurrence of genetic diversity of Frankia in the nodules sampled. DNA extracts of individual lobes that gave distinct BOX-PCR fingerprints were also amplified by PCR using primers directed against conserved regions of the 16S ribosomal RNA gene. The nucleotide sequences of the PCR products were determined and aligned with the corresponding region from other taxa for phylogenetic analysis. The sequences from Ceanothus nodules share a common ancestor to that of the Elaeagnus –infective strains.     

ARDRA对植物根瘤内共生放线菌Frankia多样性的研究

应用原核生物16SrDNA特异性引物rD1和fD1,通过ARDRA（amplified ribosomal DNA restriction analysis）法直接扩增自中国云南、东北地区赤杨属3种植物和沙棘属1种植物根瘤内FrankiaDNA,得到一长约1500bp的扩增产物,选用两种内切酶HaeⅢ、AfaⅠ联合对扩增产物进行酶切,得到稳定的酶切图谱,将所测48个感染赤杨的Frankia样本区分为3个不同的组,所测43个感染沙棘的Frankia样本区分为3个不同的组,显示根瘤内Frankia存在丰富的遗传多样性。     

Molecular phylogeny of the actinorhizal Hamamelidae and relationships with host promiscuity towards Frankia

Several of the most studied actinorhizal symbioses involve associations between host plants in the subclass Hamamelidae of the dicots and actinomycetes of the genus Frankia. These actinorhizal plants comprise eight genera distributed among three families of ‘higher’ Hamamelidae, the Betulaceae, Myricaceae, and Casuarinaceae. Contrasting promiscuity towards Frankia is encountered among the different actinorhizal members of these families, and a better assessment of the evolutionary history of these actinorhizal taxa could help to understand the observed contrasts and their implications for the ecology and evolution of the actinorhizal symbiosis. Complete DNA sequences of the chloroplast gene coding for the large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) were obtained from taxa representative of these families and the Fagaceae. The phylogenetic relationships among and within these families were estimated using parsimony and distance-matrix approaches. All families appeared monophyletic. The Myricaceae appeared to derive first before the Betulaceae and the Casuarinaceae. In the Casuarinaceae, the genus Gymnostoma derived before the genera Casuarina and Allocasuarina, which were found closely related. The analysis of character-state changes in promiscuity along the consensus tree topology suggested a strong relationship between the evolutionary history of host plants and their promiscuity toward Frankia. Indeed, the actinorhizal taxa that diverged more recently in this group of plants were shown to be susceptible to a narrower spectrum of Frankia strains. The results also suggest that the ancestor of this group of plant was highly promiscuous, and that evolution has proceeded toward narrower promiscuity and greater specialization. These results imply that a tight relationship between the phytogenies of both symbiotic partners should not be expected, and that host promiscuity is likely to be a key determinant in the establishment of an effective symbiosis.     

Identification of atypical Frankia strains by fatty acid analysis

Abstract: Ineffective, non-infective actinomycetous isolates obtained from actinorhizal nodules of Coriaria nepalensis and Datisca cannabina were identified as Frankia using whole cell fatty acid analysis. The isolates exhibited fatty-acid patterns very similar to those of confirmed Frankia strains from other host plants ( Alnus, Casuarina, Colletia, Comptonia, Elaeagnus and Hippophae ). All Frankia strains, including Coriaria and Datisca isolates, showed fatty-acid profiles very distinct from those of other actinomycetes used as controls ( Actinomyces, Geodermatophilus, Nocardia, Mycobacterium and Streptomyces ). For the genus Frankia , a characteristic pattern of five fatty acids (15:0; 15:1; 16:0 iso; 17:0 and 17:1) was found. These fatty acids comprised 75% or more of the total content. All Frankia strains could be placed into three subgroups. Coriaria isolates were found in the largest subgroup which contained most Frankia strains from other hosts while ineffective strains from Alnus, Elaeagnus and Datisca were distributed in all three subgroups of Frankia .     

Isolation and characterization of Frankia from nodules of actinorhizal plants of Pakistan

Frankia strains have been isolated from actinorhizal nodules of Alnus (2 strains), Casuarina (5 strains), Coriaria (1 strain), Datisca (3 strains), Elaeagnus (1 strain) and Hippophae (1 strain). The isolates were characterized for their growth on various carbon and nitrogen sources, nitrogen-fining ability in culture and nodulation of seedlings of the original host plant.     

Genetic heterogeneity among Frankia isolates from root nodules of individual actinorhizal plants

Abstract Genetic variations among selected Frankia isolates from nitrogen-fixing root nodules harvested from an individual actinorhizal plant ( Elaeagnus angustifolia L. or Shepherdia argentea Nutt.) were estimated by restriction fragment analysis of their total genomic DNA. The presence of plasmids and their restriction enzyme patterns were used as additional criteria. Certain isolates from separate nodules on the same plant were found indistinguishable, being probably clones of the same strain. An endophytic passage of a strain isolated from S. argentea on another host plant, Hippophaë rhamnoides L., did not modify the structural characteristics of the genome in the reisolates obtained. However, in some cases, especially when restriction endonucleases cleaving Frankia DNA into relatively small fragments were used, multiple infection of the actinorhizal plants with different Frankia strains and the presence of more than one strain in a nodule were demonstrated. Some aspects of variability in natural populations of Frankia are discussed.     

Saprophytic growth of inoculated Frankia sp. in soil microcosms

The potential of two Frankia strains to grow saprophytically was studied in nonsterile soil microcosms with ground leaf litter of Alnus glutinosa as the sole carbon and nitrogen sources. Strains Ag45/Mut15 and ArI3, which represent two taxonomic subgroups within the Alnus host infection group were inoculated alone, or together to investigate potential competition. Their growth was analyzed by in situ and dot-blot hybridization. A significant increase in cell numbers and filament length was observed during the first 6 weeks after inoculation for strain Ag45/Mut15, both alone and in mixed culture with strain ArI3, followed by a decrease until the end of the study after 12 weeks. The number of filaments remained unchanged. In contrast, the cell numbers and filament length of strain ArI3 were reduced significantly during the first 2 weeks and were undetectable for the remainder of the study. These results were comparable with those obtained in sterile mineral medium amended with leaf litter of A. glutinosa, although reductions in cell numbers and filament length were less pronounced than in soil microcosms. In concomitant control studies without leaf litter amendments for both experimental setups, filaments of both strains could only be detected immediately after inoculation. These results were matched in all experimental setups by concomitant shifts in the rRNA content of both strains, i.e., an immediate decline in the rRNA content for strain ArI3 after inoculation, and an increase in the rRNA content, followed by a late decline during incubation for strain Ag45/Mut15. These results demonstrated that Frankia strain Ag45/Mut15 could grow saprophytically in soil with complex carbon and nitrogen sources such as leaf litter, while the growth of strain ArI3 was not supported.     

The acetylene reduction assay inactivates root nodule uptake hydrogenase in some actinorhizal plants

Actinorhizal nodules do not usually evolve H₂ due to the action of an uptake hydrogenase. We have found that nodules of several Frankia symbioses evolved large amounts of H₂ gas when returned to air following exposure to 10 kPa C₂HT₂ during an acetylene reduction assay. Increased H₂ evolution in air persisted for several days when intact root systems of Alnus incana (L.) Moench (inoculated with Frankia UGL 011101) were treated with 10 kPa C.H₂ for 1 h. Full recovery of uptake hydrogenase activity required 4 to 8 days. Studies with crude homogenates of nodules of the same plants showed that hydrogenase (measured amperometrically with phenazine metho-sulfate as electron acceptor) was directly affected, since activity in treated nodules was only 10% of that in untreated nodules. A survey of actinorhizal symbioses revealed variation in the effect of an acetylene reduction assay on hydrogen metabolism. Nodules of three species, including Alnus rubra Bong, inoculated with Frankia HFPArD. showed complete inactivation of hydrogenase. H₂ evolution in air was 25% of the C₂H₂ reduction rate and H, evolution in Ar/O₂ was equal to the QH₂ reduction rate. Two symbioses, Ceanothus americanus L. (soil inoculant) and Batista glomerata Baill. (soil inoculant) showed no change following an acetylene reduction assay. A third group of symbioses showed an intermediate response.     

Studies of Seedling Inoculation of Frankia in Elaeagnus mollis Diels

The research on the preparation of Frankia solution, seedling culture and inoculation was carried out. Results indicated that the collective amount of cells may be increased when the logarithm phase cells were used as the inoculum and regularly homogenizing and transplanting inoculum by the method of magnetic stirring were adopted during the cultural process of Frankia. The inoculative tests verified that the strain with the highest ability to infect Elaeagnus mollis Diels is SIB 1301118 isolated from Elaeagnus angustifolia L. other than SIB 1332281 isolated from Elaeagnus mollis Diels. The effectivities of inoculation for germinating seeds and the seedlings to be transplanted were seed soaking and root soaking in Frankia solution preparel respectively. Early inoculation was beneficial to the growth and nodulation of seedings. The fefectivity of late inoculation was bad. Sand-soil mixture (2:1) was properly suitable to the seed germination and seedling growth. The seedling growth and nodulative rate could be promoted when 1/4 Sileris-Yong nutrient solution was suppplimented regularly.     

Nitrogenase activity decay and energy supply in Frankia after addition of ammonium to the host plant Alnus incana

A clone of Alnus incana (L.) Moench was grown in symbiosis with a local source of Frankia or with Frankia Ar14. Seven to 9-week-old plants were given 20 m M NH₄Cl (20 m M KCl = control) for 3 days. Nitrogenase activity of intact plants decreased gradually within the 3 days of treatment to about 10% of the initial rates. Hydrogen evolution in air and total nitrogenase activity responded similarly to the treatment. Relative efficiency of nitrogenase thus remained the same throughout the study period. Control plants were not affected. Measurements of nitrogenase activity in root nodule homogenates (in vitro measurements) indicated loss of active nitrogenase rather than shortage of energy for nitrogenase activity in Frankia from ammonium-treated plants. Shoots were exposed to ¹⁴CO₂ and translocation of ¹⁴C to Frankia vesicle clusters prepared from root nodules was studied. Frankia vesicle clusters from ammonium-treated plants contained about half as much ¹⁴C as those of control plants during all 3 days studied. One explanation for the observed effects is that a reduced supply of carbon to Frankia vesicles in the root nodules caused a reduced metabolic rate, including reduced protein synthesis and synthesis of nitrogenase.     

Total and CO-reactive heme content of actinorhizal nodules and the roots of some non-nodulated plants

Summary The concentration of total and CO-reactive heme was measured in actinorhizal nodules from six different genera. This gave the upper limit to hemoglobin concentration in these nodules. Quantitative extraction of CO-reactive heme was achieved under anaerobic conditions in a buffer equilibrated with CO and containing Triton X-100. The concentration of CO-reactive heme in nodules of Casuarina and Myrica was approximately half of that found in legume nodules, whereas in Comptonia, Alnus and Ceanothus the concentrations of heme were about 10 times lower than in legume nodules. There was no detectable CO-reactive heme in Datisca nodules, but low concentrations were detected in roots of all non-nodulating plants examined, includingZea mays. Difference spectra of CO treated minus dithionite-reduced extracts displayed similar wavelengths of maximal and minimal light absorption for all extracts, and were consistent with those of a hemoglobin. The concentration of CO-reactive heme was not correlated to the degree to which CO inhibited nitrogenase activity nor was it affected by reducing the oxygen concentration in the rooting zone. However, there was a positive correlation between heme concentration and suberization or lignification of the walls of infected host cells. These observations demonstrate that, unlike legume nodules, high concentrations of heme or hemoglobin are not needed for active nitrogen fixation in most actinorhizal nodules. Nonetheless, a significant amount of CO-reactive heme is found in the nodules of Alnus, Comptonia, and Ceanothus, and in the roots ofZea mays. The identity and function of this heme is unknown.