Glasshouse evaluation of the growth ofAlnus rubra andAlnus glutinosa on peat and acid brown earth soils when inoculated with four sources ofFrankia

The effects of soil type (an acid peat and 2 acid brown earths) andFrankia source (3 spore-positive crushed nodule inocula and spore-negative crushed nodules containing the singleFrankia ArI5) on nodulation, N content and growth ofAlnus glutinosa andA. rubra were determined in a glasshouse pot experiment of two years duration. Plants on all soils required additional P for growth. Growth of both species was very poor on peat withA. glutinosa superior toA. rubra. The former species was also superior toA. rubra on an acid brown earth with low pH and low P content. Some plant-inoculum combinations were of notable effectivity on particular soils but soil type was the major source of variation in plant weight. Inoculation with crushed nodules containingFrankia ArI5 only gave poor infection of the host plant, suggesting that inoculation with locally-collected crushed nodules can be a preferred alternative to inoculation withFrankia isolates of untested effectivity. Evidence of adaptation ofFrankia to particular soils was obtained. Thus, while the growth of all strains was stimulated by mineral soil extracts, inhibitory effects of peat extracts were more apparent with isolates from nodules from mineral soils than from peat, suggesting that survival ofFrankia on peat may be improved by strain selection.     

Effects of soil acidity on nodulation ofAlnus glutinosa and viability ofFrankia

Summary Black alder seedlings were grown from seed for 7 weeks in six soils limed to various pH levels and inoculated withFrankia in two inoculation-seeding time combinations (inoculated and seeded concurrently; inoculated then seeded 5 weeks after inoculation). Three mine soils and three non-mine soils were used. Soil pHs in the study ranged from 3.6 to 7.6. In the second inoculation-seeding time combination, a series of soil samples at each of the pH levels below 7.0 were relimed to pH 7.0 immediately prior to seeding. The purpose of the study was to examine the effects of soil acidity on the nodulation of black alder byFrankia and the viability ofFrankia in acid soils. Based on the average number of nodules established per seedling, soil pH was determined to be a significant factor affecting nodulation in the mine soils. The highest levels of nodulation occurred between soil pH 5.5 and 7.2. Below pH 5.5, nodulation was reduced. There was also evidence of decreased viability of the endophyte below pH 4.5.     

Distribution of spore-positive and spore-negative nodules ofAlnus incana ssp.rugosa in Maine,USA

The distribution of spore-positive (sp+) and spore-negative (sp−) root nodules ofAlnus incana ssp.rugosa (DuRoi) Clausen (speckled alder) was examined at 29 sites with a wide range of environmental conditions in Maine, USA. These included: pH 3.4 to 7.0, soil texture ranging from coarse gravel to clay to organic soils, elevation from 3 to 591 m and latitude 43 to 47°N. Habitat types included disturbed areas, streamsides, swamps and old fields. Sp (−) nodules were substantially more common, making up 76% of all nodules, whereas only 24% were sp (+). Sp (−) nodules often occurred in pure stands and predominated at disturbed sites with mineral soils at the surface and in old fields and swamps with pH>4.0 Sp (+) nodules were nearly always found in mixture with sp (−) nodules. They occurred primarily at streamside and lakeshore sites where they made up 40% of the nodules and at sites with pH<4.0 regardless of habitat type. It is suggested that sp (−) strains ofFrankia may be maintained at a site by saprophytic growth in soil and thus nodulate newly established hosts, whereas sp (+) strains may be maintained primarily by spore production within nodules and thus depend on extended presence of the host.     

Survival ofFrankia strains introduced into soil

Factors affecting the establishment of Alnus/Frankia symbioses were studied partly by following the survival ofFrankia strains exposed to different soil conditions, and partly by investigating the effect of pH on nodulation. TwoFrankia strains were used, both of the Sp⁻ type (sporangia not formed in nodules). One of the strains sporulated heavily, while the other formed mainly hyphae. The strains originated fromAlnus incana root nodules growing in soils of pH 3.5 and 5.0. The optimum pH for their growth in pure culture was found to be 6.7 and 6.2, respectively. The strains were introduced into twoFrankia-free soils, peat and fine sand. Their survival, measured as the persistance of nodulation capacity using the plant infection technique, was followed for 14 months. The survival curves of the strains were similar despite the morphological differences between the strains in pure culture. The nodulation capacities declined over time both at 14 and 22°C. Survival was better in soils limed to a pH above 6 than in soils at their original pH (peat 2.9, fine sand 4.2). The effect of pH on nodule formation in Alnus seedlings by theFrankia strains was studied in liquid culture. The number of nodules increased linearly within the pH range studied (3.5–5.8). No nodules were formed at pH 3.5.     

Morphological and molecular characterization of Frankia sp. isolates from nodules of Alnus nepalensis Don

Nodules collected from Alnus nepalensis growing in mixed forest stands at three different sites around Shillong, were crushed in various culture media to obtain isolates of Frankia. The isolates were found to have typical Frankia morphology as revealed by the scanning electron microscope. Seedlings inoculated with isolates or crushed nodules formed nitrogen fixing nodules. Frankia specific DNA probes amplified the DNA of the tested isolate AnpUS4. Partial nucleotide sequence of the 16S rRNA gene indicated that AnpUS4 was phylogenetically distinct from all other Frankia strains characterized so far.     

Frankia inoculation,soil biota,and host tissue amendment influence Casuarina nodulation capacity of a tropical soil

The effects of soil biota, Frankia inoculation and tissue amendment on nodulation capacity of a soil was investigated in a factorial study using bulked soil from beneath a Casuarina cunninghamiana tree and bioassays with C. cunninghamiana seedlings as capture plants. Nodulation capacities were determined from soils incubated in sterile jars at 21 °C for 1, 7, and 28 days, after receiving all combinations of the following treatments: ± steam pasteurization, ± inoculation with Frankia isolate CjI82001, and ± amendment with different concentrations of Casuarina cladode extracts. Soil respiration within sealed containers was determined periodically during the incubation period as a measure of overall microbial activity. Soil respiration, and thus overall microbial activity, was positively correlated with increasing concentrations of Casuarina cladode extracts. The nodulation capacity of soils inoculated with Frankia strain Cj82001 decreased over time, while those of unpasteurized soils without inoculation either increased or remained unaffected. The mean nodulation capacity of unpasteurized soil inoculated with Frankia CjI82001 was two to three times greater than the sum of values for unpasteurized and inoculated pasteurized soils. Our results suggest a positive synergism between soil biota as a whole and Frankia inoculum with respect to host infection.     

Early colonization of red alder and Douglas fir by ectomycorrhizal fungi and Frankia in soils from the Oregon coast range

The potential for mycorrhizal formation and Frankia nodulation were studied in soils from six sites in the Pacific Northwest. The sites included young and old alder stands, a 1-year-old conifer clear-cut, a young conifer plantation, and rotation-aged and old-growth conifer stands. A bioassay procedure was used with both red alder and Douglas fir seedlings as hosts. After 6 weeks growth, seedlings of both hosts were harvested every 3 weeks for 21 weeks and numbers of nodules and ectomycorrhizal types estimated. Nodules formed on red alder and ectomycorrhizae formed on both alder and Douglas fir in soil from all sites. Nodulation potential was highest in soil from the alder stands and the conifer plantation. Seven morphologically distinct ectomycorrhizal types were recovered on Douglas fir and five on alder. Only Thelephora terrestris, a broad-host-range mycobiont, formed mycorrhizae on both hosts. New ectomycorrhizal types formed on both hosts throughout the bioassay. Ectomycorrhizal colonization of alder was greatest in the alder and clear-cut soils. Low ectomycorrhizal colonization on alder was found in soils from sites where conifers were actively growing. Ectomycorrhizal colonization of Douglas fir was highest in the young alder and conifer plantation soils and was low in the rotation-aged conifer soil. The highest diversity of ectomycorrhizal types was found on alder in the conifer clear-cut soil and on Douglas fir in the rotation-aged conifer soil. Effects of host specificity, nodulation and mycorrhiza-forming potential and nodule-mycorrhiza interactions on seedling establishment are discussed in relation to seral stage dynamics and attributes of pioneer ectomycorrhizal fungal species.     

The production and utilisation of monoclonal antibodies for identification of a Frankia strain utilised as inoculum for Casuarina equisetifolia

Monoclonal antibodies were raised against Frankia 0RS020607, a strain isolated originally by H.G. Diem from nodules of Casuarina equisetifolia from Senegal. One of these antibodies, mAb8C5, was shown by ELISA to have high, but not absolute specificity for 0RS020607. This antibody was employed to investigate the mobility and persistence of 0RS020607 in plantations of C. equisetifolia. Seedlings were inoculated in pots of sand in a forest nursery with 0RS020607, with local crushed nodule suspensions or were left uninoculated. They were planted out after 5 months in experimental plots on a moderately fertile black soil site and on a low organic, oxidised red soil site. Compared with crushed nodule inoculated seedlings or uninoculated controls, growth of seedlings at transplant was improved by inoculation with Frankia 0RS020607. However, 4 years after transplant to experimental plots, the growth of trees receiving different treatments was similar. The possibility that movement of ORS 020607 between treatment plots contributed to new nodulation and enhanced growth of uninoculated trees was tested using mAb8C5 in ELISA of Frankia mycelium, extracted from the nodules of trees of the three treatments. No significant differences in reactivity were detected between nodules from uninoculated and 0RS020607 inoculated trees at either the black or the red soil sites, showing that 0RS020607 moved between treatment plots at both sites. However, at both sites, nodules from plots of trees that were inoculated originally with local crushed nodules gave reactions in ELISA that were significantly lower than values for 0RS020607 inoculated trees, possibly due to the competitive effects for new nodulation of enhancement of the indigenous population of Frankia. Serological techniques using antibodies of high specificity against Frankia strains have value for rapid screening of field samples as a preliminary for further analysis by more discriminatory techniques based on assays of genetic polymorphisms.     

Diversity of frankiae in root nodules of Morella pensylvanica grown in soils from five continents

Bioassays with Morella pensylvanica as capture plant and comparative sequence analyses of nifH gene fragments of Frankia populations in nodules formed were used to investigate the diversity of Frankia in soils over a broad geographic range, i.e., from sites in five continents (Africa, Europe, Asia, North America, and South America). Phylogenetic analyses of 522-bp nifH gene fragments of 100 uncultured frankiae from root nodules of M. pensylvanica and of 58 Frankia strains resulted in a clear differentiation between frankiae of the Elaeagnus and the Alnus host infection groups, with sequences from each group found in all soils and the assignment of all sequences to four and five clusters within these groups, respectively. All clusters were formed or dominated by frankiae obtained from one or two soils with single sequences occasionally present from frankiae of other soils. Variation within a cluster was generally low for sequences representing frankiae in nodules induced by the same soil, but large between sequences of frankiae originating from different soils. Three clusters, one within the Elaeagnus and two within the Alnus host infection groups, were represented entirely by uncultured frankiae with no sequences from cultured relatives available. These results demonstrate large differences in nodule-forming frankiae in five soils from a broad geographic range, but low diversity of nodule-forming Frankia populations within any of these soils.     

A modified sucrose fractionation procedure for the isolation of frankiae from actinorhizal root nodules and soil samples

Summary The isolation and pure culture of the symbiotic nitrogen-fixing frankiae has always been difficult. In the past the isolation of these actinomycetes directly from soil samples has proven impossible and isolations from root nodules of many genera has been only poorly successful. We report here a modified sucrose fractionation procedure which increased the success of isolations from root nodules and which permitted the isolation ofFrankia directly from soil samples. Crushed nodule suspensions or soil suspensions were incubated briefly in 0.7% phenol (carbolic acid) just before application to a sucrose density gradient. This phenol incubation decreased the number of contaminating eubacteria and fungi but more importantly increased the number ofFrankia developing on the isolation plates. If the phenol incubation was used solely without sucrose fractionation noFrankia were isolated, suggesting the death of the organisms due to phenol toxicity. The use of selective nitrogen-deficient media proved important for the isolation of frankiae from soils.     

Effects of Zn<Superscript>2+</Superscript> on nodulation and growth of a South American actinorhizal plant, <Emphasis Type="Italic">Discaria americana</Emphasis> (Rhamnaceae)

Discaria americana is a xerophytic shrub which lives in symbiosis with an actinomycete of the genus Frankia. The objective of this paper was to investigate the effects of high soil Zn²⁺ concentrations on growth and nodulation on the association Discaria americana–Frankia with the aim of determining if this association is suitable for improving contaminated soils. Two experiments were performed in 1 dm³ pots containing soil and different Zn additions, from 0 to 2,000 mg Zn²⁺ kg⁻¹ dry soil, with or without N fertilization. Zn additions strongly delayed shoot and root growth, but once growth was initiated, the biomass production of the plants supplied with moderate Zn amounts did not differ from the control plants. Zn reduced the final nodule number, but not the total nodule biomass. At the end of the experiment only the highest Zn treatments showed a lower nodule weight than the control plants, while N addition completely inhibited nodulation. It is concluded than Zn reduces the number of Frankia infections, but once the actinomycete is inside the roots, nodules can continue growing according to plant demand for N, compensating the reduced nodule number with more biomass. On the other hand, there is a toxic effect of Zn itself on plants when present in very high concentrations.     

Frankia and Alnus rubra Canopy Roots: An Assessment of Genetic Diversity, Propagule Availability, and Effects on Soil Nitrogen

The ecological importance of microbial symbioses in terrestrial soils is widely recognized, but their role in soils that accumulate in forest canopies is almost entirely unknown. To address this gap, this study investigated the Frankia–Alnus rubra symbiosis in canopy and forest floor roots at Olympic National Park, WA, USA. Sixteen mature A. rubra trees were surveyed and Frankia genetic diversity in canopy and forest floor nodules was assessed with sequence-based nifH analyses. A seedling bioassay experiment was conducted to determine Frankia propagule availability in canopy and forest floor soils. Total soil nitrogen from both environments was also quantified. Nodules were present in the canopies of nine of the 16 trees sampled. Across the study area, Frankia canopy and forest floor assemblages were similar, with both habitats containing the same two genotypes. The composition of forest floor and canopy genotypes on the same tree was not always identical, however, suggesting that dispersal was not a strictly local phenomenon. Frankia seedling colonization was similar in canopy soils regardless of the presence of nodules as well as in forest floor soils, indicating that dispersal was not likely to be a major limiting factor. The total soil nitrogen of canopy soils was higher than that of forest floor soils, but the presence of Frankia nodules in canopy soils did not significantly alter soil nitrogen levels. Overall, this study indicates that the Frankia–A. rubra symbiosis is similar in canopy and forest floor environments. Because canopy roots are exposed to different environmental conditions within very small spatial areas and because those areas can be easily manipulated (e.g., fertilizer or watering treatments), they present microbial ecologists with a unique arena to examine root–microbe interactions.     

基于高通量测序分析西藏沙棘根瘤内生菌的多样性

根瘤是微生物侵染植物根部并与之形成的共生结构,这些微生物都可被称为植物内生菌。豆科植物根瘤中的内生菌常常又被称为根瘤菌,而侵染非豆科植物形成根瘤的主要是放线菌弗兰克氏菌,这些非豆科植物又被称为放线菌结瘤植物。西藏沙棘是一种典型的放线菌结瘤植物,由于其分布生境的特殊性,对其根瘤内生菌的研究具有重要的生态意义。对于西藏沙棘根瘤内生菌的研究,培养方法因难以模拟自然条件而不易获得纯培养,高通量测序技术对其多样性的研究提供了便利。因此,本研究以生长在甘肃省天祝县金强河河滩地的西藏沙棘根瘤为材料,采用16S rRNA基因扩增子高通量测序方法,结合OTU分析,对西藏沙棘根瘤内生菌的多样性进行探讨。实验结果表明,西藏沙棘根瘤内生菌具有丰富的多样性,根瘤内的优势属为共生固氮的弗兰克氏菌属（Frankia）,其相对丰度为47.63%,共检测到7个弗兰克氏菌属的OTUs;根瘤内除弗兰克氏菌外,还存在大量的非弗兰克氏菌,共检测到1523个OTUs,隶属于22个门、33个纲、69个目、113个科和202个属,相对丰度排名前9的属中有25个非弗兰克氏菌属的OTUs。该研究也表明,西藏沙棘根瘤内生菌具有丰富的多样性,西藏沙棘根瘤中不仅存在着可共生固氮的弗兰克氏菌,并且还分布着非弗兰克氏菌;在同一根瘤样品中,弗兰克氏菌属还具有不同的物种。本研究不仅拓展了西藏沙棘根瘤内生菌多样性的研究方法,还为同一寄主植物中弗兰克氏菌多样性的研究提供了分析思路。     

Nodulation potential of soils from red alder stands covering a wide age range

Red alder (Alnus rubra Bong.) stands in the Pacific Northwest are the common first stage in succession following disturbance. These stands are highly productive and contribute a large amount of N to the soils as a result of their N₂-fixing symbiosis with Frankia. As these alder stands age, the soils not only increase in total N, but concentrations of NO^– ₃ increase and pH decreases as a result of nitrification. The objective of this study was to determine how the nodulation capacity of Frankia varies as red alder stands age and if differences in nodulation capacity are related to changes in soil properties. Nodulation capacity was determined by a red alder seedling bioassay for soils from red alder stands in the Oregon coast range covering a wide range of ages. Six chronosequences were sampled, each containing a young, an intermediate, and an older alder stand. Soil total N, total C, NO^– ₃, NH⁺ ₄, and pH were measured on the same soil samples. These factors as well as alder stand characteristics were compared with nodulation capacity in an attempt to identify soil characteristics typical in developing alder stands that most strongly affect nodulation capacity. Soil pH and NO^– ₃ concentration were highly correlated with nodulation capacity and with each other. Cluster analysis of the sites using these two variables identified two groups with distinctly different nodulation capacities. The cluster with the higher nodulation capacity was lower in NO^– ₃ and higher in pH than the other cluster, which included the majority of sites. There was substantial overlap in the age ranges for the two clusters and there was no significant correlation between age and nodulation capacity. Thus nodulation capacity appears to be most closely related to soil properties than to stand age.     

The capacity of Jamaican mine spoils, agricultural and forest soils to nodulate Myrica cerifera, Leucaena leucocephala and Casuarina cunninghamiana

Soils from seven sites on the island of Jamaica were assayed for the symbiotic diazotrophs Frankia and Rhizobium using serial dilutions. Most probable number and least squares regression methods were used to estimate each soil's capacity to nodulate native Myrica cerifera, exotic Leucaena leucocephala and exotic Casuarina cunninghamiana. The sample sites included a montane forest, a slash-and-burn agricultural site, reclaimed bauxite mining areas, abandoned sugar cane fields, and a garden plot. None of the host plants used in the bioassay were present on the sites sampled except for scattered L. leucocephala on one site. Frankia capable of nodulating M. cerifera, which is native to Jamaican highlands, occurred at all sites sampled. No C. cunninghamiana-infective Frankia was detected in soils sampled. Only soils from one site on the tropical coastal plain harbored rhizobia able to nodulate L. leucocephala (37 nodulation units cm^?3 of soil). A subset of nodulated M. cerifera and L. leucocephala reduced acetylene to ethylene indicating nitrogenase activity. The slash-and-burn agricultural site, which was situated at an elevation of 200 m and possessed both high natural fertility and high soil moisture-supplying capacity, had significantly greater Myrica infectious capacity (1 000 nodulation units cm^?3 of soil) than the other sites (7?207 nodulation units cm^?3 of soil). A planned, paired comparison revealed that a recently cultivated sugar cane field and a recently reclaimed bauxite mining site together had significantly less Myrica-infective Frankia (4 nodulation units cm^?3 of soil) than a corresponding pair of sites consisting of a sugar cane field abandoned for 25 years and a bauxite mining site reclaimed 20 years before sampling (118 nodulation units cm^?3 of soil). Results indicate that Myrica-infective Frankia is widespread in Jamaica, that the number of Myrica-infective Frankia units vary from site to site in accordance with soil type and soil history, that Jamaican sites sampled lack soil Frankia populations capable of nodulating a casuarina host, that rhizobial symbionts capable of nodulating L. leucocephala may be geographically restricted to lowlands in Jamaica, and that the occurrence of Frankia in these soils is independent of host plant presence.     

Evolutionary implications of nucleotide sequence relatedness between Alnus nepalensis and Alnus glutinosa and also between corresponding Frankia microsymbionts

Frankia DNAs were isolated directly from root nodules of Alnus nepalensis and Alnus nitida collected from various natural sites in India. For comparison, a nodule sample from Alnus glutinosa was also collected from Tuebingen, Germany. Nucleotide sequence analyses of amplified 16S–23S ITS region revealed that one of the microsymbionts from Alnus nepalensis was closely related to the microsymbiont from Alnus glutinosa. A similar exercise on the host was also carried out. It was found that one sample of Alnus nepalensis was closely related to Alnus glutinosa sequence from Europe. Since both Frankia and the host sequences studied revealed proximity between Alnus glutinosa and Alnus nepalensis, it is hypothesised that the common progenitor of all the alders first entered into an association with Frankia, and the symbiotic association has evolved since.     

Natural Diversity of Nodular Microsymbionts of Alnus glutinosa in the Tormes River Basin

The genetic diversity of Frankia strains nodulating Alnus glutinosa along the basin of the Tormes River was studied on DNA extracted directly from nodules. Frankia strains inhabiting root nodules at 12 different locations, ranging in altitude from 409 to 1181 m, were characterized. For that, we amplified the whole IGS region between 16S–23S rDNA and performed a restriction fragment length polymorphism (RFLP) analysis with four restriction enzymes. Two different RFLP patterns (termed A and B) were obtained with HaeIII, indicating the existence of two different groups of Frankia strains. Three different nodule extracts from each of the two RFLP groups were selected for further analyses. Sequencing of the 16S–23S rDNA IGS showed a 100% of intragroup homology and also confirmed the difference (98.4% level of similarity) between the Frankia strains in the two nodule extract groups. The phylogenetic analyses based on the two 16S–23S rDNA IGS sequences obtained in this study and other previously published sequences indicated that Frankia strains TFAg5 and TFAg23 (chosen as representative of HaeIII–RFLP group A and B, respectively) are quite similar to other strains nodulating plants of A. rhombifolia and A. viridis in California (pairwise levels of similarity including gaps ranged from 97.8% to 98.6%), together with which they form a single group. To put the Frankia strains representative of each HaeIII–RFLP group in the context of overall Frankia diversity we amplified and sequenced the 16S rDNA and glnII gene from nodular DNA. An also remarkable fact found in this study was that Frankia strains belonging to the HaeIII–RFLP group A were distributed all along the river course, from the lowest site sampled to the highest, while Frankia strains placed into RFLP group B were restricted to the upper Tormes River, being exclusively found at altitudes of 946 m or higher.     

Evaluation of strain competitiveness in Rhizobium leguminosarum bv phaseoli using a nod + fix− natural mutant

Competition from native soil rhizobia is likely to be an important factor limiting Phaseolus vulgaris L. inoculant response in Latin America. We used UMR 1116, a nod ⁺ fix^– natural mutant of Rhizobium leguminosarum bv phaseoli strain CC511, as a reference strain to study competition for nodulation sites in this species. When P. vulgaris cv Carioca was planted in soils containing different proportions of UMR 1116 and the effective and competitive strain UMR 1899, UMR 1116 occupied more than 50% of the nodules at all inoculant ratios tested, though increasing the proportion of UMR 1899 in the inoculant did enhance the number and percentage of effective nodules and plant dry weight. Sixty two strains of bean rhizobia were tested in competition with UMR 1116. An inoculant ratio of 1:1 was used, with all strains applied to the soil rather than to seeds. Strains varied in the number and percentage of effective nodules produced in competition with UMR 1116, and in plant dry weight, and there was a strong correlation between variation in each of these traits and plant N accumulation. Seven of the strains (UMR 1073, 1084, 1102, 1125, 1165, 1378 and 1384) were identified as both superior in competitive ability and active in N₂ fixation. Site of placement of the inoculant and ambient temperature influenced strain response.Journal paper 16736, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

Are Northwestern Patagonian “mallín” wetland meadows reservoirs of <Emphasis Type="BoldItalic">Ochetophila trinervis</Emphasis> infective <Emphasis Type="BoldItalic">Frankia?</Emphasis>

“Mallín” (plural mallines) is a particular kind of wetland occurring in Patagonian steppe and forests. In Northwest Patagonia, mallines are humid meadows with high net primary production. It was previously found that a mallín soil in the steppe devoid of actinorhizal plants had a higher Frankia nodulation capacity in Ochetophila trinervis (sin. Discaria trinervis) than other soils in the region. Under the hypothesis that mallín wetland meadows are reservoir of infective Frankia, we studied the Frankia nodulation capacity in O. trinervis of 12 mallín and their neighbouring steppe soils, by using plant bioassays. A qualitative plant bioassay showed that infective Frankia was present in most soils. The number of nodules per plant in seedlings inoculated with mallín soils was negatively correlated with soil water content while the opposite was true for plants inoculated with soils from neighbouring steppe. A quantitative bioassay was performed with eight representative soils, selected according to the number of nodules per plant produced in the qualitative assay and to the presence or not of different actinorhizal plants at the sites. Frankia nodulation units per cm³ of soil (NU) in mallín soils were higher than those in steppe. Water and organic matter content of soils were correlated with the higher nodulation capacity of mallines, which may account for the saprotrophic growth of Frankia in soils. The symbiosis was effective in plants inoculated with all soil samples. These results suggest that Northwestern Patagonian mallín wetland meadows are reservoirs of infective and effective Frankia propagules in O. trinervis.     

Characterization of genetic diversity ofFrankia strains in nodules ofAlnus nepalensis (D. Don) from the Hengduan Mountains on the basis of PCR-RFLP analysis of thenifD-nifK IGS

Nodule samples from 90A. nepalensis individuals were collected at five sites in the Hengduan Mountains. PCR-RFLP analysis of IGS betweennifD andnifK genes was directly applied to unculturedFrankia strains in the nodules. Sizes of thenifD-nifK IGS amplicons and genetic distance between the RFLP patterns from these samples were noticeably different, indicating significant genetic variation in theFrankia population. There were some nodule samples, which produced more than one PCR fragment, and compound RFLP patterns, indicating thatFrankia strains with different PCR-RFLP patterns coexisted in the same host plant under natural conditions. Among the 29 restriction patterns obtained, 5 patterns were found in more than one population and occurred in the majority of samples, while each of the other 24 patterns were represented by only one or two samples and were endemic to a particular population. From the calculatedGst and UPGMA cluster analysis, genetic diversity ofFrankia strains was inferred to be related to climate and glaciation history in the Hengduan Mountains.