Toxicity and accumulation of thallium in bacteria and yeast

Thallium sulphate inhibited microbial growth, withBacillus megaterium KM, more sensitive to the metal thanSaccharomyces cerevisiae andEscherichia coli. Inhibition ofB. megaterium KM andS. cerevisiae, but not ofE. coli, was alleviated by increasing the potassium concentration of the medium; inhibition of respiration ofS. cerevisiae, but not ofE. coli, was similarly alleviated. Thallium was rapidly bound, presumably to cell surfaces, byS. cerevisiae andE. coli, and was progressively accumulated by energy-dependent transport systems (probably concerned primarily with potassium uptake) with both organisms. Thallium uptake kinetics suggested more than one transport system operated in yeast, possibly reflecting a multiplicity of potassium transport systems. ApparentK _m andK _i values for competitive inhibition of thallium uptake by potassium indicatedS. cerevisiae to have a higher affinity for thallium uptake than for potassium, whileE. coli had a transport system with a higher affinity for potassium than for thallium. The likely systems for thallium transport are discussed. A mutant ofE. coli with tenfold decreased sensitivity to thallium was isolated and apparently effected surface binding of thallium in amounts equivalent to the wild type organism, but showed no subsequent uptake and accumulation of the metal from buffer, even though it was able to accumulate potassium to normal intracellular concentrations during growth. Abbreviations: Metal are referred to by their recognised atomic symbols (e.g. TI = Thallium; K = potassium; Co = cobalt)     

Assessment of genomic and species relationships in Triticum and Aegilops by PAGE and by differential staining of seed albumins and globulins

Summary Endosperm protein components from common bread wheats (Triticum aestivum L.) and related species were extracted with aluminum lactate, pH 3.2, and examined by electrophoresis in the same buffer. Electrophoretic patterns of the albumins and globulins were compared to evaluate the possibility that a particular species might have contributed its genome to tetraploid or hexaploid wheat. Together with protein component mobilities, differential band staining with Coomassie Brilliant Blue R250 was employed to test the identity or non-identity of bands. Eight species and 63 accessions, representative of Triticum and Aegilops were tested. Considerable intraspecific variation was observed for patterns of diploid but not for tetraploid or hexaploid species. Patterns of some accessions of Triticum urartu agreed closely with major parts of the patterns of Triticum dicoccoides and T. aestivum. A fast-moving, green band was found in all accessions of T. urartu and of Triticum boeoticum, however, that was not found in those of T. dicoccoides or T. aestivum. This band was present in all accessions of Triticum araraticum and Triticum zhukovskyi. Patterns of Aegilops longissima, which has been suggested as the donor of the B genome, differed substantially from those of T. dicoccoides and T. aestivum. Finally, two marker proteins of intermediate mobility were also observed and may be used to discriminate between accessions of T. araraticum/T. zhukovskyi and those of T. dicoccoides/T. aestivum.     

Intermolecular and intramolecular transposition and transposition immunity in Tn3 and Tn2660

Summary Intermolecular transposition of Tn2660 into pCR1 was measured at 30°C in recA ^– and recA ⁺ hosts as between 2.6 and 5.5x10^–3, a similar value to that previously found for Tn3. No cointegrate structures were found under conditions where 10⁴ transposition events occurred. Immunity to intermolecular transposition of Tn2660, similar to that found for Tn3 was demonstrated by showing that the above transposition frequency was reduced by a factor of between 10^–3 and 10^–4 when a mutant Tn2660 (resulting in the synthesis of a temperaturesensitive -lactamase) was present in the recipient plasmid. Intramolecular transposition of Tn3 was found to occur under the same conditions as previously demonstrated for Tn2660 giving rise to similar end products, in which the newly introduced Tn3 is oriented inversely to the resident Tn3 and the DNA sequence between the two transposons has been inverted. Thus, in all respects functional identity of the transposition activities of Tn3 and Tn2660 is shown, thereby identifying characteristics of intramolecular transposition that are not readily accommodated by current models of transposition.     

Phylogeny of the Celastreae (Celastraceae) and the relationships of Catha edulis (qat) inferred from morphological characters and nuclear and plastid genes

The phylogeny of Celastraceae tribe Celastreae, which includes about 350 species of trees and shrubs in 15 genera, was inferred in a simultaneous analysis of morphological characters together with nuclear (ITS and 26S rDNA) and plastid (matK, trnL-F) genes. A strong correlation was found between the geography of the species sampled and their inferred relationships. Species of Maytenus and Gymnosporia from different regions were resolved as polyphyletic groups. Maytenus was resolved in three lineages (New World, African, and Austral-Pacific), while Gymnosporia was resolved in two lineages (New World and Old World). Putterlickia was resolved as nested within the Old World Gymnosporia. Catha edulis (qat, khat) was resolved as sister to the clade of Allocassine, Cassine, Lauridia, and Maurocenia. Gymnosporia cassinoides, which is reportedly chewed as a stimulant in the Canary Islands, was resolved as a derived member of Gymnosporia and is more closely related to Lydenburgia and Putterlickia than it is to Catha. Therefore, all eight of these genera are candidates for containing cathinone- and/or cathine-related alkaloids.     

Flexibility of Pollinator-Flower Relationship in Isodon umbrosus and I. effusus (Lamiaceae) and its Relation to Fruit-set and Seed-set

Isodon umbrosus was pollinated by two bumblebee pollinators, Bombus honshuensis and B. diversus. B. diversus was a secondary pollinator when both pollinators were present, but in the absence of B. honshuensis, B. diversus pollinated I. umbrosus, and the resulting seed production was comparable to when visited by the two bumblebee species together. Not only the high visitation frequency of B. diversus but also the low ratio of seed to ovules (= average seed set × fruit set) of I. umbrosus compensated for the low pollination efficiency of B. diversus. Since, the low pollination efficiency of B. diversus, based on the finding that the low average seed set of I. umbrosus did not require a definite morphological match between flowers and pollinators, and did not require very frequent visitations to each flower. In other words, Isodon umbrosus could briefly maintain constant seed production under the condition of the low ratio of seeds to ovules (the low seed set and the low fruit set) and a large number of flowers without B. honshuensis as legitimate pollinator. On the other hand, I. effusus was exclusively pollinated by B. diversus and produced almost the same volume of seeds as I. umbrosus. Isodon effusus had fewer flowers than I. umbrosus, but this smaller number of flowers was compensated for by the high ratio of seeds to ovules in I. effusus, which was maintained by frequent visitations of the morphologically matched pollinator, B. diversus. Received 17 September 1999/ Accepted in revised form 10 February 2000     

The oxidation and reduction of pyridine nucleotides by Rhodopseudomonas spheroides and Chlorobium thiosulfatophilum

Summary The light-induced formation of NADH by whole cells of Rhodopseudomonas spheroides has been followed fluorimetrically and found to lag slightly behind cytochrome c oxidation. The uncoupler, FCCP¹, abolished NADH formation which was also inhibited by HOQNO¹. Electron flow from NADH to oxygen or cytochrome c was inhibited in chromatophores of R. spheroides by HOQNO, antimycin A and rotenone. From the known properties of the inhibitors used it is deduced that NADH formation in the light is dependent upon reversed electron flow. No light-induced formation of NAD(P)H by whole cells or chromatophores of Chlorobium thiosulfatophilum was detected either fluorimetrically or by extraction followed by enzymic assay although cytochrome c oxidation was extensive in whole cells. Extracts of C. thiosulfatophilum catalysed the rapid reduction of endogenous or mammalian cytochrome c; unlike R. spheroides this activity was found almost entirely in the soluble fraction and was insensitive to HOQNO, antimycin A and rotenone. No cytochrome b was detected in C. thiosulfatophilum by difference spectroscopy of pyridine haemochromes of acetone powders. The K _m for NADH of NADH-cytochrome c reductase in both organisms was about 3 mol; the reductase was inhibited by NAD. The rates of NADPH-cytochrome c reductase in R. spheroides particles were too low for K _m determination; for C. thiosulfatophilum particles the K _m for NADPH was about 300 mol. The addition of NADH to soluble extracts of either organism caused the reduction of endogenous flavin that was reoxidised by ferricyanide. The NADH-cytochrome c reductase of C. thiosulfatophilum was not separated from ferredoxin on a DEAE column. It is concluded that in C. thiosulfatophilum the formation of NADH in an energy-linked reaction is unlikely; the possibility of a cyclic electron flow involving chlorophyll, ferredoxin, flavoprotein and cytochrome c is discussed.     

Activation and inhibition of mitochondrial adenosine triphosphatase by various anions and other agents

The activating or inhibiting actions of a variety of anion species and of oligomycin, aurovertin and Dio-9 on the ATPase of a sonic particle preparation of rat liver mitochondria have been characterized by measurements of the relevantV _max,K _i andK _m values.The normalV _max was increased by a factor near 7 by the anions: dichromate, chromate, pyrophosphate, orthophosphate, orthoarsenate and sulphate. The fully activating concentration varied from about 2 mM for dichromate to 150 mM for sulphate. The increase inV _max was accompanied by a time-dependent decrease in (K _i)ADP, but there was no change in (K _m)ATP. The increase inV _max by the activating anions was abolished by aurovertin; but in presence of oligomycin, the lowV _max was increased by the activating anions by the same factor as theV _max in absence of oligomycin.Certain anions, notably azide, decreasedV _max, but did not affect (K _i)ADP or (K _m)ATP. The decrease inV _max by azide and oligomycin were approximately additive. Even at high concentration, Dio-9 was without detectable effect on the ATPase, but it had a gramicidinlike effect on the intact mitochondria.The specificity of the ATPase for ATP relative to GTP was found to be attributable to the high value of (V _max)ATP compared with (V _max)GTP. The values of (K _m)ATP and (K _m)GTP were virtually the same.Some rationalization of these and other supporting observations is attempted in terms of present knowledge of the constitution of the ATPase complex.     

云南地区鸭源沙门菌的分离鉴定及耐药性和毒力基因分析

【背景】沙门菌是一种重要的食源性人兽共患病原菌,可引起多种食源性疾病。【目的】了解云南地区鸭源沙门菌病的流行现状、耐药现象及毒力基因携带等基本情况。【方法】无菌采集云南各地区病死鸭肝脏样品169份进行沙门菌分离,对分离株进行血清分型鉴定、药敏及相关耐药基因、毒力基因筛查。【结果】分离到鸭源沙门菌48株,分离率为28.40%,鉴定出3种血清型,其中肠炎沙门菌为优势血清型。分离株对青霉素G、林可霉素、克林霉素和利福平的耐药率达100%,每株菌至少对3类6种及以上的抗生素耐药,单株最高可耐14种,产生了22种耐药谱型。共检出耐药基因5种,bla_TEM和tetB检出率分别为27.08%和22.92%,tetA、sul2和EBC的检出率较低。毒力基因共检出10种,其中,SPI-1(avrA)、SPI-3(mgtC)、SPI-4(siiD)、SPI-5(sopB)和bcfC检出率均高达100%,SPI-2(ssaQ)、spvB、spvC、pefA和stn的检出率均达60%以上,cdtB未检出。【结论】云南地区鸭源沙门菌主要流行血清型为肠炎沙门菌,耐药性及多重耐药情况严重,耐药机制复杂,耐药基因与耐药表型符合率低,毒力基因检出率较高。研究结果可为云南地区鸭群沙门菌病的防控和净化提供参考。     

The growth and EPA synthesis ofShewanella oneidensis MR-1 and expectation of EPA biosynthetic pathway

Shewanella oneidensis MR-1 has the ability to inhale certain metals and chemical compounds and exhale these materials in an altered state; as a result, this microorganism has been widely applied in bioremediation protocols. However, the relevant characteristics of cell growth and biosynthesis of PuFAs have yet to be thoroughly investigated. Therefore, in this study, we have attempted to characterize the growth and fatty acid profiles ofS. oneidensis MR-1 under a variety of temperature conditions. The fastest growth ofS. oneidensis MR-1 was observed at 30°C, with a specific growth rate and doubling time of 0.6885 h⁻¹ and 1.007 h. The maximum cell mass of this microorganism was elicited at a temperature of 4°C. The eicosapentaenoic acid (EPA) synthesis ofS. oneidensis MR-1 was evaluated under these different culture temperatures.S. oneidensis MR-1 was found not to synthesize EPA at temperatures in excess of 30°C, but was shown to synthesize EPA at temperatures below 30°C. The EPA content was found to increase with decreases in temperature. We then evaluated the EPA biosynthetic pathway, using a phylogenetic tree predicted on 16s rRNA sequences, and the homology of ORFs betweenS. oneidensis MR-1 andShewanella putrefaciens SCRC-2738, which is known to harbor a polyketide synthase (PKS)-like module. The phylogenetic tree revealed that MR-1 was very closely related to bothMoritella sp., which is known to synthesize DHA via a PKS-like pathway, andS. putrefaciens, which has been reported to synthesize EPA via an identical pathway. The homology between the PKS-like module ofS. putrefaciens SCRC-2738 and the entire genome ofS. oneidensis MR-1 was also analyzed, in order to mine the genes associated with the PKS-like pathway inS. oneidensis MR-1. A putative PKS-like module for EPA biosynthesis was verified by this analysis, and was also corroborated by the experimental finding thatS. oneidensis MR-1 was able to synthesize EPA without the expression of dihomo-γ-linoleic acid (DGLA) and arachidonic acid (AA) formed during EPA synthesis via the FAS pathway.     

Phenotypic and genotypic characterization of tetracycline and minocycline resistance in Clostridium perfringens

The aim of this study was to determine the incidence of tetracycline resistance and the prevalence of tetracycline-resistance genes in strains of Clostridium perfringens isolated from different sources between 1994 and 2005. Susceptibility to tetracycline and minocycline in strains from humans (35 isolates), chickens (15 isolates), food (21 isolates), soil (16 isolates) and veterinary sources (6 isolates) was determined, and tetracycline-resistance genes were detected. Resistance was most common in strains isolated from chickens, followed by those from soils, clinical samples and foods. The most highly resistant strains were found among clinical and food isolates. tetA(P) was the most common resistance gene, and along with tetB(P) was found in all resistant strains and some sensitive strains. One tetracycline-resistant food isolate had an intact tet(M) gene. However, PCR fragments of 0.4 or 0.8 kb with high degrees of identity to parts of the tet(M) sequences of other bacteria were found, mainly in clinical isolates, and often in isolates with tetB(P). No correlation between level of sensitivity to tetracycline or minocycline and the presence of tetA(P), tetB(P) or part of tet(M) was found. The presence of part of tet(M) in some strains of C. perfringens containing tetB(P) may have occurred by recent gene transfer.     

Comparisons of recruitment, survival, and growth in invasive and native saplings on a volcano

We monitored the recruitment, survival, and growth of tree saplings on invasive (Larix kaempferi) versus native species (Betula and Populus) using 16 20 m × 20 m plots established along elevation gradient on the volcano Mount Koma, Japan, for 7 years because the sapling behaviors should determine forest structures. The crowding of overstory consists mostly of Larix decreased with increasing elevation. Larix recruits were conspicuous, particularly at middle elevation where overstory crowding was intermediate, while Betula recruits were least. Larix overstory crowding inhibited the recruitment of all the taxa, although intermediate crowding promoted the recruitment of Larix. The restriction of sapling emergence was conspicuous at lower elevation where the overstory crowding was highest, probably because of shading, and/or competition with overstory trees. Sapling recruitment for all taxa was restricted at higher elevation, due to high stresses derived from direct solar radiation and strong wind without overstory. The survival of saplings was 96% for Larix and Betula, while it was ca. 50% for Populus. Larix overstory decreased the survival and growth of all the taxa, except Larix survival and Betula growth. The results implied that Larix could establish by high survival once the recruits succeeded everywhere and native sapling regeneration was restricted by Larix overstory. Strong recruitment, survival, and growth of Larix, together with resistance to overstory crowding, enables it to dominate and persist in such disturbed areas regardless of the canopy closure.     

The biosynthesis of taurine fromN-acetyl-l-cysteine and other precursorsin vivo and in rat hepatocytes

Summary The synthesis of taurine fromN-acetylcysteine has been examined in ratsin vivo and in rat hepatocyte suspensionsin vitro. In ratsin vivo, administration ofN-acetylcysteine significantly increased urinary taurine (3 fold) 24h after dosing and liver glutathione levels. Liver taurine was not increased significantly. In hepatocytes incubated in the presence ofN-acetylcysteine, glutathione concentration increased to a maximum after 1 hour but the increase was not dependent on the concentration ofN-acetylcysteine. In contrast, after an initial lag phase, taurine synthesis increased in relation to the concentration ofN-acetylcysteine and continued for 3 hours. Glutathione synthesis seems to be preferential to taurine synthesis. Taurine synthesis from cysteine sulphinate was greater and from hypotaurine was greatest and maximal after 1 hour. Implications for the mechanism of protection byN-acetylcysteine are discussed.     

Cloning and characterization of the Azotobacter vinelandii recA gene and construction of a recA deletion mutant

Summary The recA gene of Azotobacter vinelandii was isolated from a genomic library by heterologous complementation of an Escherichia coli recA mutation for resistance to UV radiation. The A. vinelandii recA gene was localized on adjacent PstI fragments of 1.3 and 1.7 kb. The cloned A. vinelandii recA gene was functionally analogous to the E. coli recA gene. It was also able to complement the E. coli recA mutation for homologous recombination. A recA deletion mutant of A. vinelandii was constructed. This mutant was sensitive to DNA-damaging agents like UV rays, methyl methane sulfonate (MMS) and nalidixic acid and was deficient in homologous recombination.     

Processing and characteristics of soybean-fortified gari

Two cassava cultivars were fermented for 3 days with the incorporation of soybean residue or full-fat flour at 25% (w/w) to produce gari. Unfortified gari was superior in taste (P<0.05) to the soybean-fortified gari, though consumers still accepted the latter. Fortified samples from two of the three procedures used, in which soybean was added at different stages of the fermentation, were rated similarly (P>0.05). The water activity of soybean-fortified gari was <0.7, the swelling capacity was 2.5 to 3.3, pH was 5.1±0.2 and total cyanide was 0.8 to 1.3 mg/100 g. The crude protein content of soybean-fortified gari was 8 to 12% (w/w), much higher than the 1 to 2% (w/w) of the unfortified gari. Soybean-fortified gari is recommended for use in areas where cassava is consumed and protein intake is inadequate.The authors are with the Biology Department, The Polytechnic, Ibadan, Nigeria.     

Heat production and body temperature during cooling and rewarming in overweight and lean men

Objective: To compare overweight and lean subjects with respect to thermogenesis and physiological insulation in response to mild cold and rewarming. Research Methods and Procedures: Ten overweight men (mean BMI, 29.2 ± 2.8 kg/m²) and 10 lean men (mean BMI, 21.1 ± 2.0 kg/m²) were exposed to cold air for 1 hour, followed by 1 hour of rewarming. Body composition was determined by hydrodensitometry and deuterium dilution. Heat production and body temperatures were measured continuously by indirect calorimetry and thermistors, respectively. Muscle activity was recorded using electromyography. Results: In both groups, heat production increased significantly during cooling (lean, p = 0.004; overweight, p = 0.006). The increase was larger in the lean group compared with the overweight group (p = 0.04). During rewarming, heat production returned to baseline in the overweight group and stayed higher compared with baseline in the lean group (p = 0.003). The difference in heat production between rewarming and baseline was larger in the lean (p = 0.01) than in the overweight subjects. Weighted body temperature of both groups decreased during cold exposure (lean, p = 0.002; overweight, p < 0.001) and did not return to baseline during rewarming. Discussion: Overweight subjects showed a blunted mild cold‐induced thermogenesis. The insulative cold response was not different among the groups. The energy‐efficient response of the overweight subjects can have consequences for energy balance in the long term. The results support the concept of a dynamic heat regulation model instead of temperature regulation around a fixed set point.     

Suppression by Zygorrhynchus moelleri of Growth and Sclerotium Production by Rhizoctonia solani and Sclerotinia sclerotiorum

As indicated by reduced cellulolysis, Zygorrhynchus moelleri suppressed mycelial growth in Rhizoctonia solani and Sclerotinia sclerotiorum. Sclerotium production by both pathogenic fungi was also reduced by Z. moelleri in dual sand-oatmeal cultures. The viability of sclerotia produced by S. sclerotiorum, but not those produced by R. solani, was greatly reduced. Sclerotium production by S. sclerotiorum on celery and tomato segments was reduced to a much greater extent when Z. moelleri was applied to the plant tissue 24 h before the pathogen than when applied at the same time or 24 h after the pathogen.     

Renal function and organic anion and cation transport during dehydration and/or food restriction in chickens

The effect of dehydration in the presence or absence of continued food intake on renal function was evaluated in chickens. In addition, renal transport of organic anions and cations under these conditions was assessed in vitro by uptake of ¹⁴C-para-aminohippuric acid and ¹⁴C-tetraethylammonium bromide by renal slices. Water restriction with continued food intake resulted in increases in serum osmolality and serum concentrations of sodium, uric acid, calcium and total protein. If food was restricted in addition to water, only serum osmolality and sodium concentration were significantly increased after 48 hours. Dehydration with continued access to food resulted in marked decreases in extracellular fluid volume, glomerular filtration rate and effective renal plasma flow. If food was restricted during dehydration, the decrease in effective renal plasma flow was attenuated despite reductions in glomerular filtration rate and extracellular fluid comparable to that seen in dehydrated birds allowed free access to food. Transport of organic anions was significantly increased after 24 and 48 hours of water restriction, regardless of whether food was withheld. Enhanced transport of organic anions in the presence of decreased glomerular filtration rate and effective renal plasma flow during dehydration may promote precipitation of urates and nephrosis in chickens.Abbreviations cpm counts per minute - dpm disintegrations per minute - ECF extracellular fluid - ERPF effective renal plasma flow - GFR glomerular filtration rate - PAH para-aminohippuric acid - SEM standard error of the mean - TEA tetraethylammonium bromide     

Temperature- and moisture-induced changes in the structure of the nematode fauna of a semiarid grassland –- patterns and mechanisms

A field experiment was established to examine the effects of temperature and moisture modifications on the nematode fauna of a semiarid grassland. Several combinations of drying, wetting, warming and cooling were applied to plots and compared with untreated control plots. The experiment was performed from July to October 1996. A significant shift was observed in the structure of the nematode fauna between late summer and early autumn. This shift was manifested in the disappearance of four rare genera; Ecumenicus, Eucephalobus, Paraphelenchus and Pungentus. A significant decrease was found in the density of Acrobeles, Aphelenchoides, Ditylenchus and Prismatolaimus; in addition there was a significant increase in the density of Cephalobus, Helicotylenchus, Paratylenchus and Tylenchorhynchus. Community structural change was represented by an initial decrease in nematode generic richness of 30–50%, and in a statistically significant decrease of nematode diversity in the control and all treated plots. Thereafter, emergence of a new community was demonstrated. Data show that temperature manipulation was the main factor to influence nematode diversity, Maturity Index, and Plant Parasite Index. However, nematode population density was influenced predominantly by the soil moisture content. Coenological analysis of soil nematode fauna appears to be a useful tool for the biological monitoring of the effects of global change on semiarid grasslands.     

Chemical Profiling and Assessment of Antineurodegenerative and Antioxidant Properties of Veronica teucrium L. and Veronica jacquinii Baumg.

Neuroprotective potential of V. teucrium and V. jacquinii methanol extracts was analyzed. Chemical analysis of investigated extracts showed the presence of phenolic acid derivatives, flavonoids and one secoiridoid. The detected flavonoids derived from flavones (luteolin and isoscutellarein in V. jacquinii ; apigenin, isoscutellarein and luteolin in V. teucrium ) and flavonol (quercetin in V. jacquinii ). Acteoside was the dominant compound in V. jacquinii , while plantamajoside and isoscutellarein 7‐O ‐(6?‐O ‐acetyl)‐β ‐allosyl (1?→2?)‐β ‐glucoside were the major phenolics in V. teucrium . Additionally, the antineurodegenerative activity was tested at concentrations of 25, 50, and 100 μg/ml using acetylcholinesterase (AC hE) and tyrosinase (TYR ) assays. The inhibition of both enzymes was achieved with the investigated extracts, ranging from 22.78 to 35.40% for AC hE and from 9.57 to 16.38% for TYR . There was no statistical difference between the activities of the analyzed extracts. Our data indicate that V. teucrium and V. jacquinii may have beneficial effects against Alzheimer 's and Parkinson 's disease.     

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.