几种非豆科植物根瘤内生菌侵染特征的研究

自不同科、属、种的非豆科植物根瘤分离内生菌,对其寄主植物进行了交叉侵染,结果表明,这些Frankia菌对不同寄主的侵染没有明显的专一性,供试菌可以进行跨越科、属、种的侵染,但有的菌株对于某些植物的侵染,可能存在一些特殊情况,相同菌株对不同植物的侵染能力,以及不同菌株对同一寄主的侵染能力是有差异的。从同一种植物根瘤中分离的不同菌株,侵染能力也有高低之分,供试菌随寄主植物的改变,侵染能力及所建立的共生系统固氮活性有所降低,侵染原寄主植物所形成的根瘤固氮活性较高的菌株,在改变寄主后所形成的根瘤固氮活性也比较高,在一定条件下,寄主植物的结瘤量与根瘤固氮活性呈正相关,而侵染不同寄主后,根瘤中菌体孢子的表面结构也发生了一定变化。     

Effect of inoculation and leaf litter amendment on establishment of nodule-forming Frankia populations in soil

High-N(2)-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [(15)N]NO(3) and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N(2) fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N(2) fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% +/- 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% +/- 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% +/- 6%) than by group IV (81% +/- 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N(2) fixation rates by (15)N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N(2)-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N(2)-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

Natural diversity of Frankia strains in actinorhizal root nodules from promiscuous hosts in the family Myricaceae.

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N(2)-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.     

Biology of Frankia strains, actinomycete symbionts of actinorhizal plants.

Frankia strains are N2-fixing actinomycetes whose isolation and cultivation were first reported in 1978. They induce N2-fixing root nodules on diverse nonleguminous (actinorhizal) plants that are important in ecological successions and in land reclamation and remediation. The genus Frankia encompasses a diverse group of soil actinomycetes that have in common the formation of multilocular sporangia, filamentous growth, and nitrogenase-containing vesicles enveloped in multilaminated lipid envelopes. The relatively constant morphology of vesicles in culture is modified by plant interactions in symbiosis to give a diverse array of vesicles shapes. Recent studies of the genetics and molecular genetics of these organisms have begun to provide new insights into higher-plant-bacterium interactions that lead to productive N2-fixing symbioses. Sufficient information about the relationship of Frankia strains to other bacteria, and to each other, is now available to warrant the creation of some species based on phenotypic and genetic criteria.     

Recent advances in the biogeography and genecology of symbiotic Frankia and its host plants

Molecular phylogenetic approaches have begun to outline the origin, distribution and diversity of actinorhizal partners. Geographic isolation of Frankia and its host plants resulting from shifting continents and dispersal patterns have apparently led to the development of Frankia genotypes with differing affinities for host genera, even within the same plant family. Actinorhizal plant genera of widespread global distribution tend to nodulate readily even outside their native ranges. These taxa may maintain infective Frankia populations of considerable diversity on a broad scale. Arid environments seem to have distinctive actinorhizal partnerships, with smaller and more specific sets of Frankia symbionts. This has led to the hypothesis that some host families have taxa that are evolving towards narrow strain specificity, perhaps because of drier habitats where fewer Frankia strains would be able to survive. Harsh conditions such as water-saturated soils near lakes, swamps or bogs that are typically acidic and low in oxygen may similarly lessen the diversity of Frankia strains present in the soil, perhaps limiting the pool of frankiae available for infection locally and, at a larger scale, for natural selection of symbiotic partnerships with host plants. Recent molecular ecological studies have also provided examples of Frankia strain sorting by soil environment within higher order cluster groupings of Frankia host specificity. Future frontiers for ecological research on Frankia and actinorhizal plants include the soil ecosystem and the genome of Frankia and its 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.     

Assessing the phylogeny of Frankia-actinorhizal plant nitrogen-fixing root nodule symbioses with Frankia 16S rRNA and glutamine synthetase gene sequences

Actinomycetes from the genus Frankia induce nitrogen-fixing root nodules on actinorhizal plants in the "core rosid" clade of eudicots. Reported here are nine partial Frankia 16S rRNA gene sequences including the first from host plants of the rosaceous genera Cercocarpus and Chamaebatia, 24 partial glutamine synthetase (GSI; glnA) sequences from Frankia in nodules of 17 of the 23 actinorhizal genera, and the partial glnA sequence of Acidothermus cellulolyticus. Phylogenetic analyses of combined Frankia 16S rDNA and glnA sequences indicate that infective strains belong to three major clades (I-III) and that Clade I strains consisting of unisolated symbionts from the Coriariaceae, Datiscaceae, Rosaceae, and Ceanothus of the Rhamnaceae are basal to the other clades. Clock-like mutation rates in glnA sequence alignments indicate that all three major Frankia clades diverged early during the emergence of eudicots in the Cretaceous period, and suggest that present-day symbioses are the result of an ancestral symbiosis that emerged before the divergence of extant actinorhizal plants.     

Low genetic diversity among Frankia spp. strains nodulating sympatric populations of actinorhizal species of Rosaceae, Ceanothus (Rhamnaceae) and Datisca glomerata (Datiscaceae) west of the Sierra Nevada (California)

Frankia spp. strains typically induce N2-fixing root nodules on actinorhizal plants. The majority of host plant taxa associated with the uncultured Group 1 Frankia strains, i.e., Ceanothus of the Rhamnaceae, Datisca glomerata (Datiscaceae), and all actinorhizal members of the Rosaceae except Dryas, are found in California. A study was conducted to determine the distribution of Frankia strains among root nodules collected from both sympatric and solitary stands of hosts. Three DNA regions were examined, the 5' end of the 16S rRNA gene, the internal transcribed spacer region between the 16S and 23S rRNA genes, and a portion of the glutamine synthetase gene (glnA). The results suggest that a narrow range of Group 1 Frankia spp. strains dominate in root nodules collected over a large area of California west of the Sierra Nevada crest with no apparent host-specificity. Comparisons with Group 2 Frankia strain diversity from Alnus and Myrica within the study range suggest that the observed low diversity is peculiar to Group 1 Frankia strains only. Factors that may account for the observed lack of genetic variability and host specificity include strain dominance over a large geographical area, current environmental selection, and (or) a past evolutionary bottleneck.     

Features of the intrageneric Alnus-Frankia specificity

Host specificity between local Frankia strains and native alders [Alnus incana (L.) Moench and A. glutinosa (L.) Gaertn.] was evaluated in inoculation experiments. Pure cultures of Frankia , whether originating from A. incana or A. glutinosa , were infective and effective on both host species. These pure cultures were isolated from spore-negative (Sp⁻) nodules. From spore-positive (Sp⁺) nodules no Frankia isolates were obtained. This strain type resisted all our isolation attempts and therefore crushed nodules had to be used for Sp⁺ type inoculations.
The Sp⁺ type Frankia populations differed in their host specificity. Sp⁺ nodules from A. glutinosa were effective on both alder species, but Sp⁺ nodules from A. incana induced effective nodules only on the original host; on A. glutinosa only small (1-3mm) prenodule-like structures were found. Such A. glutinosa plants died on N-free medium, thus showing that these nodules were ineffective. In the effective nodules the middle cortex was dominated by infected cells filled with vesicle clusters. In the ineffective nodules only a few cortical cells were infected and sporangia predominated in these cells. Surprisingly enough they also contained vesicle-like structures as demonstrated in electron micrographs.     

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.     

Foliar-applied lanthanum chloride increases growth and phosphorus acquisition in phosphorus-limited adzuki bean seedlings

Plant and Soil - Members of the nitrogen-fixing actinobacterial genus Frankia are typically isolated from root nodules and generally infective on the same plant species. Several Frankia strains...     

Protective effect of Brucella outer membrane complex-bearing liposomes against experimental murine brucellosis

Abstract Nodulation ability was tested for Frankia strains HFPCcI3 and ELI, and Frankia sources A.t. and G.a. from Allocasuarina torulosa and Gymnostoma australianum , respectively, on A. torulosa Miq., Casuarina cunninghamiana Miq., G. australianum L. Johnson and Elaeagnus triflora Roxb. It was shown that A. torulosa and C. cunninghamiana formed nodules only with the Frankia sources obtained from their own host plant, while E. triflora formed nodules with three of the four Frankia sources tested. All nodules formed were effectively fixing nitrogen. Specific nitrogenase activity was highest in E. triflora inoculated with the Frankia strain isolated from nodules of the same species. Identification of Frankia sources in the nodules was performed by use of PCR amplification of DNA with a random primer. PCR amplification of DNA isolated from nodules of G. australianum and E. triflora inoculated with Frankia strain EL1 revealed, when compared with DNA amplified from free living Frankia strain EL1, that there was only one Frankia strain causing the observed nodules.     

Effects of symbiosis with Frankia and arbuscular mycorrhizal fungus on the natural abundance of 15N in four species of Casuarina

The effect of interactions between Casuarina species, Frankia strains and AMF on nitrogen isotope fractionation within the plant were determined under conditions where changes in source nitrogen were minimized by growing plants in mineral nitrogen-deficient conditions and without added organic N. Casuarina cunninghamiana, C. equisetifolia, C. glauca, and C. junghuniana were inoculated singly with three Frankia strains or were dual inoculated with Frankia and Glomus fasciculatum. The %N and delta 15N of separated parts of plants inoculated with the three Frankia strains or with Frankia + Glomus were not significantly different within Casuarina species. However, the slow-growing C. junghuniana differed in several variables from the other three species. There was a highly significant, linear relationship between the natural logarithms of cladode N content and delta 15N of plants of the four Casuarina species when inoculated with Frankia or with Frankia + Glomus, showing that nitrogen supply and the correlated variable, plant growth rate, were major determinants of delta 15N. Provision of small quantities of (NH4)2SO4 or KNO3 increased several-fold the growth of three of the Casuarina species when inoculated with Frankia alone or with Frankia + Glomus. Within species, mycorrhizal and non-mycorrhizal plants receiving supplementary soluble phosphate were of similar dry weights at harvest. delta 15N values for cladodes of C. cunninghamiana, C. equisetifolia and C. glauca were similar, but values for the poor growing C. junghuniana were more variable and, with the exception of plants receiving KNO3, were lower than those of the other three species. Reduced growth due to suboptimal availability of N or P had a major influence on delta 15N and, in these conditions where plants could not access significant amounts of organic N, outweighed any effects on cladode delta 15N of colonization by Glomus. delta 15N values of nodules were higher than other parts of Frankia or Frankia + Glomus inoculated Casuarinas, conceivably due to retention in nodules of fixed N, with delta 15N close to zero.     

The Actinorhizal Symbiosis

Abstract The term ``actinorhiza' refers both to the filamentous bacteria Frankia, an actinomycete, and to the root location of nitrogen-fixing nodules. Actinorhizal plants are classified into four subclasses, eight families, and 25 genera comprising more than 220 species. Although ontogenically related to lateral roots, actinorhizal nodules are characterized by differentially expressed genes, supporting the idea of the uniqueness of this new organ. Two pathways for root infection have been described for compatible Frankia interactions: root hair infection or intercellular penetration. Molecular phylogeny groupings of host plants correlate with morphologic and anatomic features of actinorhizal nodules. Four clades of actinorhizal plants have been defined, whereas Frankia bacteria are classified into three major phylogenetic groups. Although the phylogenies of the symbionts are not fully congruent, a close relationship exists between plant and bacterial groups. A model for actinorhizal specificity is proposed that includes different levels or degrees of specificity of host-symbiont interactions, from fully compatible to incompatible. Intermediate, compatible, but delayed or limited interactions are also discussed. Actinorhizal plants undergo feedback regulation of symbiosis involving at least two different and consecutive signals that lead to a mechanism controlling root nodulation. These signals mediate the opening or closing of the window of susceptibility for infection and inhibit infection and nodule development in the growing root, independently of infection mechanism. The requirement for at least two molecular recognition steps in the development of actinorhizal symbioses is discussed.     

Casuarina root exudates alter the physiology, surface properties, and plant infectivity of Frankia sp. strain CcI3

The actinomycete genus Frankia forms nitrogen-fixing symbioses with 8 different families of actinorhizal plants, representing more than 200 different species. Very little is known about the initial molecular interactions between Frankia and host plants in the rhizosphere. Root exudates are important in Rhizobium-legume symbiosis, especially for initiating Nod factor synthesis. We measured differences in Frankia physiology after exposure to host aqueous root exudates to assess their effects on actinorhizal symbioses. Casuarina cunninghamiana root exudates were collected from plants under nitrogen-sufficient and -deficient conditions and tested on Frankia sp. strain CcI3. Root exudates increased the growth yield of Frankia in the presence of a carbon source, but Frankia was unable to use the root exudates as a sole carbon or energy source. Exposure to root exudates caused hyphal "curling" in Frankia cells, suggesting a chemotrophic response or surface property change. Exposure to root exudates altered Congo red dye binding, which indicated changes in the bacterial surface properties at the fatty acid level. Fourier transform infrared spectroscopy (FTIR) confirmed fatty acid changes and revealed further carbohydrate changes. Frankia cells preexposed to C. cunninghamiana root exudates for 6 days formed nodules on the host plant significantly earlier than control cells. These data support the hypothesis of early chemical signaling between actinorhizal host plants and Frankia in the rhizosphere.     

Evidence that some Frankia sp. strains are able to cross boundaries between Alnus and Elaeagnus host specificity groups.

Phenotypic and genotypic methods were used to prove the existence of Frankia strains isolated from an Elaeagnus sp. that are able to cross the inoculation barriers and infect Alnus spp. also. Repeated cycles of inoculation, nodulation, and reisolation were performed under axenic conditions. Frankia wild-type strain UFI 13270257 and three of its coisolates did exhibit complete infectivity and effectiveness on Elaeagnus spp. and Hippopha? rhamnoides and variable infectivity on Alnus spp. Microscopical observation of host plant roots showed that these strains are able to infect Alnus spp. by penetrating deformed root hairs. Reisolates obtained from nodules induced on monoxenic Alnus glutinosa, Alnus incana, and Elaeagnus angustifolia resembled the parent strains in host infectivity range, in planta and in vitro morphophysiology, isoenzymes, and nif and rrn restriction fragment length polymorphisms, thus fulfilling Koch's postulates on both host plant genera. Alnus and Elaeagnus group-specific polymerase chain reaction DNA amplifications, DNA-DNA hybridizations, and partial gene sequences coding for 16S rRNA provided evidence for the genetic uniformity of wild-type strains and their inclusion into one and the same genomic species, clearly belonging to the Elaeagnus group of Frankia species.     

Evidence that some Frankia sp. strains are able to cross boundaries between Alnus and Elaeagnus host specificity groups.

Phenotypic and genotypic methods were used to prove the existence of Frankia strains isolated from an Elaeagnus sp. that are able to cross the inoculation barriers and infect Alnus spp. also. Repeated cycles of inoculation, nodulation, and reisolation were performed under axenic conditions. Frankia wild-type strain UFI 13270257 and three of its coisolates did exhibit complete infectivity and effectiveness on Elaeagnus spp. and Hippophaë rhamnoides and variable infectivity on Alnus spp. Microscopical observation of host plant roots showed that these strains are able to infect Alnus spp. by penetrating deformed root hairs. Reisolates obtained from nodules induced on monoxenic Alnus glutinosa, Alnus incana, and Elaeagnus angustifolia resembled the parent strains in host infectivity range, in planta and in vitro morphophysiology, isoenzymes, and nif and rrn restriction fragment length polymorphisms, thus fulfilling Koch's postulates on both host plant genera. Alnus and Elaeagnus group-specific polymerase chain reaction DNA amplifications, DNA-DNA hybridizations, and partial gene sequences coding for 16S rRNA provided evidence for the genetic uniformity of wild-type strains and their inclusion into one and the same genomic species, clearly belonging to the Elaeagnus group of Frankia species.     

Diversity and distribution of Frankia strains symbiotic with Ceanothus in California

Frankia strains symbiotic with Ceanothus present an interesting opportunity to study the patterns and causes of Frankia diversity and distribution within a particular host infectivity group. We intensively sampled Frankia from nodules on Ceanothus plants along an elevational gradient in the southern Sierra Nevada of California, and we also collected nodules from a wider host taxonomic and geographic range throughout California. The two sampling scales comprised 36 samples from eight species of Ceanothus representing six of the seven major biogeographic regions in and around California. The primary objective of this study was to use a quantitative model to test the relative importance of geographic separation, host specificity, and environment in influencing the identity of Ceanothus Frankia symbionts as determined by ribosomal DNA sequence data. At both sampling scales, Frankia strains symbiotic with Ceanothus exhibited a high degree of genetic similarity. Frankia strains symbiotic with Chamaebatia (Rosaceae) were within the same clade as several Ceanothus symbionts. Results from a classification and regression tree model used to quantitatively explain Frankia phylogenetic groupings demonstrated that the only significant variable in distinguishing between phylogenetic groups at the more local sampling scale was host species. At the regional scale, Frankia phylogenetic groupings were explained by host species and the biogeographic province of sample collection. We did not find any significant correspondence between Frankia and Ceanothus phylogenies indicative of coevolution, but we concluded that the identity of Frankia strains inhabiting Ceanothus nodules may involve interactions between host species specificity and geographic isolation.     

Biogeography of symbiotic and other endophytic bacteria isolated from medicinal Glycyrrhiza species in China

A total of 159 endophytic bacteria were isolated from surface-sterilized root nodules of wild perennial Glycyrrhiza legumes growing on 40 sites in central and northwestern China. Amplified fragment length polymorphism (AFLP) genomic fingerprinting and sequencing of partial 16S rRNA genes revealed that the collection mainly consisted of Mesorhizobium, Rhizobium, Sinorhizobium, Agrobacterium and Paenibacillus species. Based on symbiotic properties with the legume hosts Glycyrrhiza uralensis and Glycyrrhiza glabra, we divided the nodulating species into true and sporadic symbionts. Five distinct Mesorhizobium groups represented true symbionts of the host plants, the majority of strains inducing N2-fixing nodules. Sporadic symbionts consisted of either species with infrequent occurrence (Rhizobium galegae, Rhizobium leguminosarum) or species with weak (Sinorhizobium meliloti, Rhizobium gallicum) or no N2 fixation ability (Rhizobium giardinii, Rhizobium cellulosilyticum, Phyllobacterium sp.). Multivariate analyses revealed that the host plant species and geographic location explained only a small part (14.4%) of the total variation in bacterial AFLP patterns, with the host plant explaining slightly more (9.9%) than geography (6.9%). However, strains isolated from G. glabra were clearly separated from those from G. uralensis, and strains obtained from central China were well separated from those originating from Xinjiang in the northwest, indicating both host preference and regional endemism.