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.     

Production,decomposition, and nitrogen dynamics ofMyrica gale litter

Summary Myrica gale litter deposition and decomposition were studied in a central Massachusetts peatland to determine the amount of N made available to the ecosystem by these processes. Leaf litter added 114–140 g biomass m^–2 annually and contained 2.12–2.59 g N m^–2 returning about 70% as much N to the ecosystem as was fixed annually byMyrica gale. During the first five years of decomposition, the leaf liter lost only 40% of its initial biomass and released only 10% of its initial N content. About 60% of its original N mass was still present when the litter reached the permanently waterlogged zone, and thus was effectively lost to the vegetation. The low decomposition rate was due primarily to the chemical content of the litter because similarly low rates were observed in an upland forest where the native litter decayed rapidly. The initial lignin content (40%) ofM. gale litter may be largely responsible for its slow decomposition in spite of its relatively high (1.69%) initial N content.M. gale litter decayed substantially more slowly and had a much higher initial lignin content than the litter of other woody N₂-fixing plants which have been examined.     

Nutrient deficiency promotes male-biased apparent sex ratios at the ramet level in the dioecious plant <Emphasis Type="Italic">Myrica gale</Emphasis> var. <Emphasis Type="Italic">tomentosa</Emphasis> in oligotrophic environments in bogs

In populations of dioecious plants, the differences in the cost of reproduction between male and female plants can promote a male-biased sex ratio. In this study, we examine the macronutrient levels in tissues of the dioecious wetland shrub Myrica gale to identify the cost of reproduction for male and female plants and to examine the effect of nutrients on the apparent sex ratio at the ramet level. We examined plants across 12 populations of M. gale inhabiting bogs and fens in Japan. For each population, we used line transects to estimate the apparent sex ratio and measured the concentrations of nitrogen (N), phosphorus (P), and potassium (K) in the leaves sampled from male and female plants and in the fruits from female plants. For five of the populations, we calculated the flowering frequency, mortality, and the recruitment rate (as the rate of clonal propagation). We found that the proportion of females was positively affected, and the male bias of sex ratios reduced, by increases in P concentration in leaves sampled from female plants. Neither mortality nor recruitment was affected by sex or by the nutrient concentration (P, K). The flowering frequency was not affected by sex or by K concentration, but decreased with decreases in the P concentration measured in leaves. This study confirmed that reproduction in M. gale is P-limited. We found no distinct differences in the flowering frequency, mortality, or recruitment rate between the male and female plants.     

Consequences of Sporangial Development for Nodule Function in Root Nodules of Comptonia peregrina and Myrica gale

Frankia sp., the actinomycetous endophyte in nitrogen-fixing actinorhizal nodules, may differentiate two forms from its hyphae: vesicles and sporangia. In root nodules of Comptonia peregrina (L.) Coult. and Myrica gale L., sporangia may be either absent or present. Nitrogenase activity and symbiotic efficiency were contrasted in spore(+) and spore(−) nodules of these two host genera. Seedlings of C. peregrina nodulated with the spore(+) inoculum showed only 60% of the nitrogenase activity and 50% of the net size of their spore(−) counterparts after 12 weeks of culture. Measurements of acetylene reduction (i.e., nitrogenase activity) were coordinated with samplings of nodules for structural studies. Significant differences in acetylene reduction rates were discernible between spore(+) and spore(−) nodules commencing 4 weeks after nodulation, concomitant with the maturation of sporangia in the nodule. Spore(+) nodules ultimately reached less than half of the rate of nitrogenase activity of spore(−) nodules. Both types of nodules evolved only small amounts of molecular hydrogen, suggesting that both were equally efficient in recycling electrons lost to the reduction of hydrogen ions by nitrogenase. Respiratory cost of nitrogen fixation, expressed as the quotient of micromole CO₂ to micromole ethylene evolved by excised nodules, was significantly greater in spore(+) than in spore(−) nodules. M. gale spore(−) nodules showed variable effectivity, though all had low CO₂ to ethylene evolution ratios. M. gale spore(+) nodules resembled C. peregrina spore(+), with low effectivity and high respiratory cost for nitrogen fixation.     

Potential production and utilisation of oil fromTrichilia SPP. (Meliaceae)

Trichilia emetica Vahl andT. dregeana Sonder are trees indigenous to Zimbabwe and are widely planted for shade in urban areas. The seed ofTrichilia contains oil concentrations as high as most oil-rich dicotyledonous seeds and is a potential source of income for rural people. The seed production of mature trees varies greatly from one year to the next, averaging 64.7 kg of fresh seed per tree annually in Harare. Fresh seed yields approximately 308 ml oil kg¹ (approximately 30%), using a simple ram press. Trichilia oil produces a good finish on wooden surfaces and would compete successfully with other commercial wood oils. No storage problems are envisaged for either seed or oil.     

The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas.     

The history of beer additives in Europe — A review

The many excavations of medieval sites during recent years have resulted in a strong increase in archaeobotanical records including species which were used as beer additives. Since the first compilation of records by the author in 1984 relating to the two main species, namelyMyrica gale andHumulus lupulus, the number of finds has quadrupled. Distribution maps of the sites with fossil occurrence of these two species are presented and this evidence is complemented by that from written sources.M. gale seems to have been used for brewing as early as the centuries immediately before and after the birth of Christ in a small area at the Rhine estuary in the northern Netherlands. During the early and high Middle Ages there are records of this plant, in what are potentially brewing contexts, across its north-west European area of natural distribution. Written sources confirm its use in brewing as early as the tenth century. The finds ofH. lupulus indicate that this species has been used in brewing from the early Middle Ages and this hypothesis is supported by documentary evidence. Cultivation of hop began around A.D. 859. In the late Medieval period, strong competition developed between both kinds of beer, which resulted in the take-over byH. lupulus in the eighteenth century. Many other herbs of secondary importance have been used to flavour beer or to prepare medicinal beers. These are mentioned in old herbals and have been compiled in this paper. These various flavouring agents, combined with the use of all available species of cereals led to a variety of beers that is unimaginable today.     

NOTES ONPOLYSPORINAVĚZDA,WITH A DESCRIPTION OF A NEW SPECIES FROM TASMANIA

The new species,Polysporina terricolaKantvilas from south-eastern Tasmania, is described and compared to other species in the genus. It is well-characterized by a range of morphological, anatomical and ecological characters, especially by the presence of oil paraphyses and oil droplets in the hymenium, features unknown in other members of the genus. Species ofPolysporinaare best separated by the following characters: size and morphology of the apothecia, size of ascospores, thickness of paraphyses and height of the hymenium. The combinationsP. ferruginea(Lettau) M. Steiner andP. pusilla(Anzi) M. Steiner are validated. A key to the taxa studied is provided.     

Two new C-methylated flavonoids from Myrica gale

From the leaves of Myrica gale 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone has been isolated. The fruits yielded 2′-hydroxy-4′,6′-dimethoxy-3′-methyldihydrochalcone. The constitutions were deduced from spectroscopic data and confirmed by synthesis.     

Natural diversity of nodular microsymbionts of Myrica rubra

Myrica is often considered a promiscuous actinorhizal genus. However, there are large differences in diversity among Myrica spp., and M. gale does not exhibit such promiscuity in its natural environment. In order to understand the diversity of nodular microsymbionts of M. rubra in natural environments and whether or not the M. rubra is a `promiscuous' host, we studied the natural diversity of nodular microsymbionts of different cultivars of M. rubra. 15 nodules from nine horticultural cultivars of M. rubra were collected in 7 sites of eastern, southeastern, central and northern part of Zhejiang province, China. Unisolated strains were compared by sequence analyses of their nifD-nifK intergenic spacers and PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing sequences of their nifD-nifK intergenic spacers and reference strains. There is a high degree of diversity among nodular Frankia symbionts of M. rubra. Frankia strains from cluster I and cluster III were found in nodules from many different cultivars of M. rubra. Furthermore, there were sometimes two strains which belong to different infective clusters of Frankia in the same nodule, and Frankia strains of cluster I were often dominant strains when there were two strains. M. rubra can thus be considered to be promiscuous in nature. Identical sequences in nodules from different plants at widely separated sites were commonly found, indicating that some strains are cosmopolitan. Geographic separation, host selectivity for Frankia symbionts and soil environment may account for the diversity of Frankia strains and differences in Frankia populations found in M. rubra nodules. Several very closely related local Frankia populations in M. rubra nodules could be distinguished from one another by our approach.     

The effect of ferrous ions,tungstate and selenite on the level of formate dehydrogenase inClostridium formicoaceticum and formate synthesis from CO2 during pyruvate fermentation

In a medium containing a trace element solution and 10^-4 M ferrous ions the growth yield ofClostridium formicoaceticum on fructose was 5.5 g of weight per l; in the absence of metal ion solution it was 1 g per l. The specific activity of methyl viologen dependent formate dehydrogenase under both conditions was 0.28 and 0.03 units per mg of protein, respectively. It could be increased to 9.75 units when the growth medium contained 10^-4 M tungstate and 10^-5 M selenite in addition. Molybdate was only about 40% as effective as tungstate. Tungstate or molybdate could not be replaced by vanadate, selenite not by sulfide. The formate dehydrogenase catalyzed also the reduction of CO₂ to formate. The highest rate of formate synthesis was observed when pyruvate served as the reductant. No pyruvate: formate exchange but rapid pyruvate: CO₂ exchange could be observed with cell-free extracts ofC. formicoaceticum. Pyruvate is fermented byC. formicoaceticum to yield up to 1.16 mole acetate per mole of pyruvate. Resting cells accumulated some formate in addition to acetate.     

Taxonomic studies of theMuscari botryoides complex in Hungary

The ploidy level and karyotype ofMuscari botryoides (s.l.) samples from Hungary (25 localities) and Romania (1 locality:locus classicus ofM. transsilvanicum) were determined. The Romanian sample proved to be diploid (2n=18), while in Hungary both diploid and tetraploid (2n=36) populations occurred. The karyotypes of all diploid populations were similar: 2 pairs of long acrocentric (one of them usually with satellites) + 3 pairs of medium-sized submetacentric-metacentric + 4 pairs of short ± metacentric chromosomes. All diploid populations in Hungary can be identified asM. transsilvanicum. There is no reason to support the taxonM. botryoides subsp.hungaricum because it does not differ from the sample collected at thelocus classicus ofM. transsilvanicum (Romania, Sibiu-Gu?teri?a) in any of the characteristics mentioned in its protologue. Its karyotype also corresponds to that ofM. transsilvanicum. Contrary to the former assumptions, the tetraploidM. botryoides is also native to Hungary. The tetraploid karyotype seems to be somewhat of a duplication of the diploid one. Morphological characters used in the identification keys are not suitable for unambiguous separation of the taxa mentioned above, though morphometric analyses revealed some quantitative differences between diploids and tetraploids. Their separation on species level can only be supported by the supposed reproductive barriers caused by different ploidy level and chorology. In HungaryM. transsilvanicum is restricted mostly to the Eupannonian Region, the Mecsek and Villány Mts.M. botryoides does not occur in the Eupannonicum, instead it inhabits the subatlantic hilly W and SW part of Hungary and the Northern Mountain Range. The latter territory (including also the Slovak localities) seems to be the easternmost extension of the area ofM. botryoides.     

The isolation,culture and infectivity of aFrankia strain fromGymnostoma papuanum (Casuarinaceae)

A strain ofFrankia was isolated fromGymnostoma papuanum(Casuarinaceae) nodules harvested from rooted cuttings which had been inoculated with a suspension of crushedCasuarina equisetifolia nodules. Designated HFPGpI1 (catalogue #HFP021801), this strain is pigmented and similar to other pigmentedFrankia strains in cultural characteristics. A previously unknown spiraled hyphal morphology was observed at very low frequency in some cultures of this strain. HFPGpI1 is infective and effective onG. papuanum but not on anyCasuarina species tested. It also infects members of the family Elaeagnaceae andMyrica gale. The host plantG. papuanum can be infected with a wide range ofFrankia isolates and thus can be considered a promiscuous host, unlike its close relatives in the genera Casuarina and Allocasuarina which are very restrictive as to which strains may nodulate them.     

Metarhizium spp. isolates from madagascar: Morphology and effect of high temperature on growth and infectivity to the migratory locust,Locusta migratoria

Five strains ofMetarhizium anisopliae (Metsch.) Sorokin and one strain ofMetarhizium flavoviride Gams &; Rozsypal originally isolated in Madagascar were studied. Measurements of conidia and, for the first time, also of blastospores produced in a liquid medium were used for species and variety determination. Blastospores ofM. flavoviride were more homogenous in their size than those ofM. anisopliae. Growth at high temperatures between 25° and 40°C showed that 4 isolates ofM. anisopliae grew at 36°C andM. flavoviride grew at 38°C. Using alternating day/night temperatures (8/16 h) the three strains tested could also tolerate 40°/25°C. In bioassays, fiveMetarhizium spp. isolates were tested against third and fourth instar larvae ofLocusta migratoria (L.) at two alternating day/night temperatures of 30°/25°C and 36°/25°C. In the cooler regime, all strains caused a mortality of 50% within 5.9 to 8.5 days (median lethal time), while in the 36°/25°C treatment only the thermophilicM. flavoviride and oneM. anisopliae strain isolated from a soil sample gave comparable results with median lethal times of 6.8 and 7.3 days, respectively.     

Life cycle and host range of the gall-forming moth,Meskea horor (Lep.: Thyrididae), and its suitability as a biological control agent forSida acuta andS. rhombifolia (Malvaceae)

The life cycle of the moth,Meskea horor Dyar and its host specificity toSida acuta andS. rhombifolia were investigated. Moths emerge early in the morning and are usually inactive during the day. Feeding by the adults in the field was not observed. However, those in the laboratory fed on flowers ofS. acuta, S. rhombifolia andHibiscus sabdariffa, and drank water or dilute honey solution. Eggs are mainly laid on the underside of leaves. Larvae ofM. horor form galls in the stems ofS. acuta andS. rhombifolia which retard plant growth and flowering. Pupation occurs within the gall. The development time from egg to adult was 192 days and the adults lived for 11 to 14 days. Forty-eight plant species were tested to determine the host range ofM. horor. Adults emerged from seven species of plants in the family Malvaceae and larvae formed galls but died before pupating on a further 17 species.M. horor is considered to have too broad a host range to be used as a biological control agent forS. acuta andS. rhombifolia.     

Karyological variation in the group ofMyosotis alpestris (boraginacea)

A total of 6 population samples ofMyosotis stenophylla Knaf, a rare species showing great ecological disjunction in its distribution, were examined to clarify the present status of its karyological variation. In order to elucidate relationships between lowland tetraploid populations ofM. stenophylla and diploid and tetraploid montane populations ofM. alpestris F.W. Schmidt, four population samples ofM. alpestris were also examined. The karyotypes of all populations ofM. alpestris s.l. studied were highly asymmetrical and heterogeneous, being composed of metacentric, submetacentric, subtelocentric and satellited acrocentric chromosomes. The karyotype formula for haploid chromosome set was established: n=x=12=6m+2sm+3st+1t^SAT. Multivariate analysis based on chromosome length and shape showed significant differences between diploid and tetraploid forms ofM. alpestris s.l. Four numerical parameters, used to characterize the karyotype ofM. stenophylla, revealed significant differences between populations on serpentine and on non-serpentine substrates. In addition, the noticeable affinity of the karyotype of non-serpentine populations to that ofM. alpestris tetraploids has been shown by means of discriminant analysis. These data suggest that the unique features of serpentine play an important role in the origin of karyotypic differentiation within populations ofM. stenophylla.     

The origin ofMentha X gracilis (Lamiaceae). I. Chromosome numbers,Fertility, and three morphological characters

Employing nine clones ofMentha arvensis and four clones ofM. spicata, 932 F, hybrids were synthesized and compared to 20 clones ofM. x gracilis. Two clones ofM. x gracilis with 60 somatic chromosomes were matched to a selected F₁ hybrid. The other 18 clones ofM. x gracilis had somatic chromosome numbers of 60, 72, 84, and 96, and while these chromosome numbers appeared in the F₁ progeny, morphological matches correlated with their correct chromosome numbers were not synthesized. The range of pollen and seed fertility, as well as the inheritance of male-sterility, leaf pubescence, and crispness, indicates that no one character can be used to identifyM. x gracilis, but all characters can be explained fromM. arvensis x M. spicata.     

Problems in the use of plant biochemistry for establishing the safety of biological control agents for weeds: TheChondrilla andEchium/heliotropium cases

Demonstration of specificity, on the one hand, of the biological control agents for the weedChondrilla juncea L. [Compositae: Cichoriae] as compared to those for the control of the weedsEchium plantagineum L. andHeliotropium europaeum L. (Tubiflorales:Boraginaceae) represents 2 extremes. In the case ofC. juncea the phytochemical data was insufficiently known to be of value and if it had existed, would have been of little value in demonstrating quarantine safety for certain of the agents. However, in the case of the 2Boraginaceae there is strong evidence that studies based on phytochemical data would have confirmed specificity.     

Identification of alkane monoxygenase genes inAcinetobacter venetianus VE-C3 and analysis of mutants impaired in diesel fuel degradation

Cells ofAcinetobacter venetianus strain VE-C3 are able to degrade diesel fuel oil by a complex mechanism requiring the formation of cell aggregates and their further adhesion to fuel oil drops. In this work the biodegradation process inA. venetianus was studied by a combination of genetic, molecular and physiological methods. PCR amplification, sequencing and Southern blot analysis ofalkM andrubA genes coding for the alkane hydroxylase and rubredoxin were carried out. Then, 22 Alk^? mutants impaired in diesel fuel degradation were obtained by nitrosoguanidine mutagenesis and characterised by i) growth on alkanes as sole carbon and energy sources, ii) modification of cell electrophoretic properties, and iii) analysis of plasmid content. Data obtained revealed that the genetic determinants for alkane degradation are located on both the chromosome and the two plasmids harboured by VE-C3 strain (pAV1 and pAV2, 11 Kbp and 15 kbp, respectively). This organization of genes coding for alkane monoxygenase complex seems to be similar to the arrangement found in Acinetobacter sp. strains ADP1 and M1, where genes are scattered through the chromosome but, as a novelty, that some genes involved in hydrocarbon degradation are plasmid borne also.