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.     

Ecological wood anatomy ofAlnus nepalensis (Betulaceae) in East Nepal

Wood anatomical characters ofAlnus nepalensis growing in East Nepal are evaluated against three non-anatomical factors: tree height, diameter at breast height (DBH), and altitude. Samples were taken from the outermost part of the trunk of five canopy trees at 11 localities between 790 and 2,740 m above sea level. Tree height ranged from 10 to 28 m, and DBH ranged from 15 to 80 cm. Altitude and tree height are correlated with all the vessel characters studied. Among wood anatomical characters, vessel characters measured from cross sections are strongly correlated with one another, and are also correlated with vessel element and fiber-tracheid length. Multiple regression analysis using non-anatomical factors as independent variables resulted in significant correlation at 1% level in all pore characters, vessel element length, perforation plate bar number, and fibertracheid length. Regression coefficients of significant regressions are usually largest for altitude. For wood structure ofAlnus nepalensis in East Nepal, 23 to 42% of the variation is affected by non-anatomical factors. The large contribution of altitude is considered to be an indirect measure of the effect of temperature.     

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.     

Identification ofArmillaria species from hokkaido by analysis of the intergenic spacer (IGS) region of ribosomal DNA using PCR-RFLP

The intergenic spacer (IGS) region, which is located between the 3′ end of 26S ribosomal DNA (rDNA) and the 5′ end of 5S rDNA, of sixArmillaria species from Hokkaido was investigated using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Restriction with onlyAlu I could distinguishA. mellea subsp.nipponica from the other species. WithAlu I andDde I,A. ostoyae andA. gallica could be distinguished from the other species. Digestion withAlu I resulted in two patterns (types A and B) ofA. singula and three patterns (types A, B, and C) ofA. jezoensis. One pattern (type B) of the former species and two patterns (types B and C) of the latter species were each different from those of the other species.Armillaria sinapina gave only oneAlu I digestion pattern, which was identical to that ofA. jezoensis (type A) andA. singula (type A). However, by digestion withDde I,A. singula (type A) could be distinguished fromA. jezoensis (type A) andA. sinapina.     

Physiology and chemical diversity ofFrankia spp. isolated from nodules ofComptonia peregrina (L.) Coult. andCeanothus americanus L.

Summary TwoFrankia spp., isolated from the nodules of the plant hostComptonia peregrina, were found to fall into two previously described physiological groups (A and B). Of five frankia isolates fromCeanothus americanus plants of the same provenance, three belonged to physiological group A and two to a novel group whose final disposition remains to be decided. The diversity in whole cell sugar chemistry, morphology and other growth characteristics of these strains is discussed.     

Diversity of Frankia strains isolated from single nodules of Alnus glutinosa

Diversity of Frankia isolates originating from lobes of single nodules collected on Alnus glutinosa root systems has been analyzed using isozyme electrophoresis method. Analysis of isozyme patterns showed no divergence among strains isolated from the same nodule. Each nodule (among 10 assayed) was inhabited by a single Frankia strain.     

Chemical soil properties under five age series ofAlnus nepalensis plantations in the Eastern Himalayas

Summary Five age series stands ofAlnus nepalensis D. Don monocultures have been selected from the Pankhasari range of the Kalimpong forest division in the Eastern Himalayas. Chemical soil propeties such as organic carbon, total nitrogen, CN ratio, available phosphorus and exchangeable cations (Ca⁺², K⁺ and Na⁺) were analysed in all these different ages of plantation stands from the surface to 1 m deep profiles to find out the long term effect ofA. nepalensis on the soil quality of these erosion vulnerable slopes.Organic carbon, available phosphorus and total nitrogen content per-hectare increased with increasing plantation stand age. Analysis of variance for nutrients showed significant variation in depth (total nitrogenP<0.001; organic carbon,P<0.001; available phosphorus,P<0.05; exchangeable Ca⁺²,P<0.01; and exchangeable K⁺,P<0.01) and between plantation stands (organic carbon,P<0.05; and available phosphorusP<0.05). High value of total nitrogen content (34.97t ha^–1) was obtained in 46 yr old stand. Soil pH is low. It increased down the depth and in older plantations. These stands with high soil total nitrogen and organic carbon content show increased fertility of the stands. The cover ofA. nepalensis in rocky, landslide and ecosion prone slopes of the Eastern Himalayas, serves to protect and improve soil quality.     

Distribution of spore-positive and spore-negative nodules in stands ofAlnus glutinosa andAlnus incana in Finland

Summary The distribution of spore positive (Sp⁺) and spore negative (Sp⁻) nodules on the two native alder species (A. incana andA. glutinosa) in Finland was investigated. Nodules were collected throughout the country from different ecosystems (forests, swamps, lake- sea- and riversides, old pastures and fields as well as from alder plantations). OnA. incana Sp⁺ nodules predominated, whereas onA. glutinosa the vast majority of the nodules were of the Sp⁻ type. Sp⁺ nodules onA. glutinosa were found only at sites where the two alder species grew close together. This distribution pattern indicates an association of nodule type with alder species, the reasons for which are discussed. Indications of saprophytic growth in the Sp⁻ strain were also found.     

Isolation of infective and effective Frankia strains from root nodules of Alnus acuminata (Betulaceae)

Two Frankia strains were isolated from root nodules of Alnus acuminata collected in the Tucumano-oranense forest, Argentina. Monosporal cultures were obtained by plating a spore suspension of each strain and isolating a single colony. The strains (named AacI and AacIII) showed branched mycelia with polymorphic sporangia and NIR-vesicles. They differed in their ability to use carbon sources: the AacI strain grew well on pyruvate, while the AacIII strain grew on mineral medium supplemented with glucose or, alternatively, with sucrose. The two strains were sensitive to oleandomycin, erythromycin, kanamycin, penicillin G, streptomycin and chloramphenicol at 5 μg/ml. The AcIII strain exhibited a moderate resistance to rifampicin, ampicillin and vancomycin. The nitrogenase activity in vitro of the strains was significantly higher in basal medium without nitrogen than that determined in the presence of ammonium chloride. Both strains were infective on seedlings of Alnus glutinosa, inducing an approximately similar percentage of nodulated plants (80%), although strain AacIII produced a higher number of nodules per plant (≤15) than strain AacI (≤6). They were also effective for nitrogen fixation in planta, determined by the acetylene reduction assay. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Correlations between the ages of Alnus host species and the genetic diversity of associated endosymbiotic Frankia strains from nodules

Nodule samples were collected from four alder species: Alnus nepalensis, A. sibirica, A. tinctoria and A. mandshurica growing in different environments on Gaoligong Mountains,Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS between nifD and nifK genes was directly applied to uncultured Frankia strains in the nodules. A total of 21 restriction patterns were obtained. The Frankia population in the nodules of A. nepalensis had the highest genetic diversity among all four Frankia populations; by contrast, the population in the nodules of A. mandshurica had the lowest degree of divergence; the ones in the nodules of A. sibirica and A. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated that Frankia strains from A. sibirica and A. tinctoria had a close genetic relationship. Frankia strains from A. nepalensis might be the ancestor of Frankia strains infecting other Alnus species. From these results and the inference of the ages of Alnus host species, it is deduced that there was a co-evolution between Alnus and its microsymbiont Frankia in China.     

Correlations between the ages of Alnus host species and the genetic diversity of associated endosymbiotic Frankia strains from nodules

Nodule samples were collected from four alder species: Alnus nepalensis, A. si-birica, A. tinctoria and A. mandshurica growing in different environments on Gaoligong Mountains, Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS between nifD and nifK genes was directly applied to uncultured Frankia strains in the nodules. A total of 21 restriction patterns were obtained. The Frankia population in the nodules of A. nepalensis had the highest genetic diversity among all four Frankia populations; by contrast, the population in the nodules of A. mandshurica had the lowest degree of divergence; the ones in the nodules of A. sibirica and A. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated that Frankia strains from A. sibirica and A. tinctoria had a close genetic relationship. Frankia strains from A. nepalensis might be the ancestor of Frankia strain     

Morphology,physiology and infectivity of twoFrankia isolates An 1 and An 2 from root nodules ofAlnus nitida

Summary Two different strains, An 1 and An 2, were obtained from root nodules ofAlnus nitida Endl., collected from one locality in the area of its natural habitat near Bahrin, District Swat, Pakistan. The light and electron microscopy of the isolates revealed the occurrence of septate and branched hyphae bearing sporangia and vesicles. The strains differed in their growth requirements, nitrogen-fixing ability and production of extracellular pigments, thus indicating the existence of more than oneFrankia strain in the same locality. In the absence of combined nitrogen in the medium strain An 1 formed vesicles and fixed N₂ (up to 200 nmol C₂H₄. mg protein^–1.h^–1), while strain An 2 under the experimental conditions formed only few vesicles and fixed N₂ at a very low rate (ca 10 nmol C₂H₄. mg protein^–1 .h^–1). The nitrogenase activity of strain An 1 was strongly affected by the O₂ concentration.Frankia An 1 and An 2 were infective and effective onA. nitida andA. glutinosa but not onDatisca cannabina andElaeagnus umbellata. Both An 1 and An 2 strains were more infective and effective onA. glutinosa thanFrankia strains AvcIl and CpI1.     

Observations on the ultrastructure ofFrankia sp. in root nodules ofDatisca cannabina L.

Summary The fine structures of the microsymbiont inside the root nodules ofDatisca cannabina have been studied by light, by transmission- and by scanning-electron microscopy. The endophyte is prokaryotic and actinomycetal in nature. The hyphae are septate and branched, diameter 0.3–0.5 m. The tips of hyphae are swollen to form electron-dense, clubshaped to filamentous vesicles, ranging in diameter: 0.4–1.4 m. The endophyte penetrates through walls of the cortial cells. The infected zone is kidney shaped and confined to one side of the acentric stele. The orientation of infection is reversed from other actinorhizae exceptCoriaria. The hyphae are near the host cell wall and vesicles are directed towards the central vacuole. Vesicles are aseptate and no collapsing of the vesicle cell wall (void area) has been observed. Vesicle clusters structures are globular with an opening at one side of the cluster. The host cell is multinucleate or contains a lobed nucleus. Groups of mitochondria are located in between the hyphae, suggesting a strong association between the host and the endophyte for energy supply and amino acid production. The consequences of the inability to separate the mitochondria from the vesicle clusters in nodule homogenates in physiological studies have been discussed.Isolated vesicles clusters showed dehydrogenase activity, indicated by the presence of formazan crystals, after incubation with NADH and NBT. Strongest reducing activity was found within the vesicles. The possible role of filamentous vesicles in nitrogen fixation has been discussed.     

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.     

长白山三种赤杨根瘤内Frankia基因多样性的REP分析

利用REP PCR方法研究了长白山不同海拔处 3种赤杨根瘤内Frankia菌基因多样性。结果表明 ,赤杨根瘤内Frankia菌存在丰富的基因多样性。宿主和海拔因素共同影响长白山赤杨属Frankia菌的基因多样性 :东北赤杨Frankia菌的宿主专一性很强 ,西伯利亚赤杨和色赤杨的Frankia菌的有较近的亲缘关系。不同海拔的同种宿主共生的Frankia菌基因类型不同 ,相同海拔的西伯利亚赤杨和色赤杨的Frankia菌出现宿主混乱现象     

Characterization of nativeFrankia strains isolated from chilean shrubs (Rhamnaceae)

Nine nativeFrankia strains were isolated from root nodules of four chilean actinorhizal plants (Rhamnaceae). The strains were designated as ChI1, ChI2, ChI3 and ChI4 fromColletia hystrix; ReI4 and ReI6 fromRetanilla ephedra; TqI12 and TqI15 fromTalguenea quinquinervis and TtI42 fromTrevoa trinervis. By scanning electron microscopy, all the strains exhibited similar actinomycetal structures: hyphae, sporangia and vesicles. The growth patterns of the isolates in BAP medium were similar. All showed a lag phase of approximately 6–7 days, then exhibited a logarithmic phase, except the ReI4 strain which seems to follow a linear growth pattern. A common feature of all the strains was a rapid loss of biomass at the end of the growth phase. All native strains grew on BAP medium supplemented with glucose. In six out of nine strains, the glucose was the best of the carbon sources tested. However, the strains differed in their ability to use other carbon sources such as arabinose, mannitol, maltose, succinate, sucrose, pyruvate, propionate and galactose. The isolates were sensitive to six antibiotics assayed (ampicillin, penicillin G, rifampicin, chloramphenicol, erythromycin and kanamycin). Using the acetylene reduction assay, the nitrogenase activity of the strains was determined. All strains grown in BAP medium lacking a combined nitrogen source were able to reduce acetylene ‘in vitro’.     

新疆地区番茄潜叶蛾遗传多样性的ISSR分析

[目的]番茄潜叶蛾已成为世界性番茄的重要害虫,对番茄产业的发展造成了严重的威胁,研究其遗传多样性有利于揭示不同地理种群的遗传变异结构。[方法]采用ISSR分子标记技术分析了20个地理种群番茄潜叶蛾的遗传多样性和遗传结构特征。[结果]15条引物扩增出137条ISSR条带,其中多态性条带占96.35%,所有个体显示了各自独特的ISSR图谱。ISSR的标记遗传多样性结果表明,20个番茄潜叶蛾地理种群遗传距离大小范围为0.0065~0.1623,遗传一致度范围为0.8502~0.9935。种群变异来源分析表明,25.36%的遗传变异来自种群间,74.64%的变异来源于种群内部。UPGMA系统发育分析结果表明,各地理种群的聚类与地理位置无较强的关联性。[结论]番茄潜叶蛾尚处在入侵早期阶段,且具备频繁入侵和多点的特征。防控上要注意加强检疫,阻绝多点入侵来源。     

Study of the contribution of the shoot and/or root ofAlnus sp. in the compatibility between the host and a Sp+ Frankia strain using a grafting technique

Two alder species,Alnus glutinosa (L.) Gaertn. andAlnus incana (L) Moench, were inoculated with a Sp⁺ Frankia homogenate obtained fromA. incana root nodules. This inoculum formed effective nodules on the original host plant and ineffective nodules onA. glutinosa. Grafts between the two alder species were made to determine which part of the plant is involved in this phenomenon. The results obtained indicate that the compatibility between Alnus andFrankia is restricted to the root system.     

Molecular diversity of <Emphasis Type="Italic">Frankia</Emphasis> from root nodules of <Emphasis Type="Italic">Hippophae salicifolia</Emphasis> D.Don found in Sikkim

Molecular diversity of Frankia was assessed directly from the root nodules of Hippophae salicifolia naturally occurring in North Sikkim. Amplicon restriction patterns (ARPs) were developed by digesting 16S-ITS-23S amplicons with RsaI. Three ARPs were detected, showing diversity among strains of Frankia that nodulate Hippophae. This was confirmed by sequencing one amplicon each for the three ARPs. Therefore, ARP can be used as a tool for screening amplicons for nucleotide sequencing.