AMPLIFICATION OF FUNGAL rDNA-ITS REGIONS FROM NON-FERTILE SPECIMENS OF THE LICHEN-FORMING GENUSPARMELIA

A method is described for the selective amplification of single mycobiont rDNA sequences from vegetative, non-ascomatal material of the lichenized thalli ofParmelia sulcataandP. saxatilis. The method includes a simple DNA extraction procedure employing rhizines, which avoids the use of liquid nitrogen disruption methods and phenol based extractions. The resulting amplified DNA is suitable for restriction enzyme analysis, and can be used to provide material for investigating population distribution and species concepts.     

Monitoring with lichens – Conductivity methods assess salt and heavy metal damage more efficiently than chlorophyll fluorescence

In the lab, we exposed three foliose lichen species, Lobaria pulmonaria, Parmelia sulcata and Xanthoria aureola, to 0, 0.01, 0.2, and 0.6 M NaCl in combinations with copper and zinc (0, 10, 100, 500 μM). High salt concentrations adversely affected the lichen membrane integrity as measured by conductivity methods, whereas the potential photosystem II efficiency (Fv/Fm) was tolerant. High light was necessary to reduce Fv/Fm in thalli exposed to salt, whereas high light did not aggravate the conductivity. The seashore species X. aureola was much more resistant to salt than the old forest species L. pulmonaria. With respect to Cu and Zn, used concentrations had no (P. sulcata, X. aureola) or small (L. pulmonaria) effects on Fv/Fm. However, both heavy metals substantially increased conductivity in all species, consistent with membrane damage. Thus, the conductivity method detected high salt, high copper and high zinc stress much more efficiently than did the chlorophyll fluorescence method. This suggests that membrane integrity of the mycobiont is more sensitive to salt and heavy metal stress than potential photosystem II efficiency of its autotrophic partners.     

Symbiotic performance,nitrogen flux and growth of lima bean (<Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L.) varieties inoculated with different indigenous strains of rhizobia

Nitrate uptake and nitrogen inclusion into amino acids were studied in the intact thallus and isolated bionts of the lichen Parmelia sulcata with the aid of mass spectroscopic tracing of heavy isotope ¹⁵N. The isolated photobiont, the green algae Trebouxia sp. did not take up nitrate, whereas the mycobiont and intact thalli were enriched in ¹⁵N when incubated with Na¹⁵NO₃. Pulse feeding experiments with intact thalli followed by separation of photobiont showed that the labelled nitrate was originally assimilated by the mycobiont and only after that was detected in the photobiont. The isolated mycobiont after pulse labeling excreted labeled compounds into the incubation medium. Amino acids were detected in the exudate. The quantities of two amino acids considerably exceeded those of the others. One was identified as alanine, the other could not yet be identified with certainty. Both of these high-quantity compounds were also much more enriched in ¹⁵N than the others. These two compounds are proposed to be the transport forms of nitrogen within the Parmelia sulcata thallus.     

THE NEW LICHEN FAMILYGLOEOHEPPIACEAEAND ITS GENERAGLOEOHEPPIA,PSEUDOPELTULAANDGUDELIA (LICHINALES)

Gloeoheppiais re-established as a separate taxon; the genus differs fromHeppiain apothecial development and thallus structure.Gloeoheppiaand the related generaGudeliaHenssen andPseudopeltulaHenssen described here are placed in the new familyGloeoheppiaceaein theLichinales. In habit, the genera ofGloeoheppiaceaeresemble species ofHeppiaandPeltula, whereas in apothecial development they correspond to some extent to theLichinaceae. The speciesGloeoheppia polysporaHenssen,Gloeoheppia rugosaHenssen,Pseudopeltula dicyanophoraHenssen,Pseudopeltula heppioidesHenssen,Pseudopeltula myriocarpaHenssen andGudelia mexicanaHenssen, are described for the first time. InGloeoheppiaceaethe thalli are ecorticate and the hyphae reticulately branched. The prototunicate asci contain eight, or up to 16, colourless and simple, occasionally two-celled spores. In species ofPseudopeltula, the apothecia are of a complex structure. The cyanobionts are small and single-celled; inP. dicyanophora‘internal cephalodia’ with an additional, filamentous cyanobiont are formed. Lectotypes are designated forEndocarpon turgidumAch. andHeppia furvaHue in Patouill.     

Effect of lunularic acid on auxin-oxidase activity

Lunulario aoid was isolated from fresh thalli ofTargionia hypophylla L. This acid was found to enhanoe the destruction of IAA by auxin-oxidase from lentil roots. Such action can explain, at least partially, its inhibitory effect on IAA-induced plant growth.     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida,strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

Inhibition ofCitrobacter freundii by lactic acid, ascorbic acid, citric acid,Thymus vulgaris extract and NaCl at 31 °C and 5 °C

Citrobacter freundii has been implicated in food spoilage and food poisoning outbreaks. This study examines the effects of some compounds (e.g. citric acid, ascorbic acid, lactic acid, sodium chloride, andThymus vulgaris extract) on growth of two strains of Citrobacter freundii at 31 °C and 5 °C. At 31 °C, lactic acid (0.2%) or ascorbic acid (0.2%) alone completely inhibited growth of the tested strains, as there was 100% reduction in growth of the strains after 24 h incubation in nutrient broth containing these compounds.Thymus vulgaris extract (0.3%) reduced the growth rate (p<0.05), the percentages of inhibition after 24 h incubation were about 60% for both strains. NaCl (5%) greatly reduced growth, the percentages of inhibition were about 84% for both strains. Combination ofT. vulgaris extract (0.3%) and NaCl (4%) together completely inhibited growth ofC. freundii species tested. Ascorbic acid (0.1%) or citric acid (0.03%) did not affect growth of the strains (p>0.05), but a lag occurred before increase in number could be observed. In chicken and fish homogenates, combination of NaCl (4%) and ascorbic acid (0.1%) reduced the growth (p < 0.05) (growth inhibition was 40%). At 5 °C, lactic acid (0.1%) alone greatly reduced the growth (p<0.05). The activity of NaCl, or ascorbic acid alone against the tested strains was greatly increased (p<0.05). ForC. freundii 4, the percentage of growth inhibition after 6 days incubation in broth containing 3% NaCl or 0.1% ascorbic acid were 88% and 72%, respectively. ForC. freundii 38, the percentage of growth inhibition after 6 days incubation in broth containing these compounds were 60% and 54%, respectively.     

Einkerniges Protonema beiBryopsis undPseudobryopsis myura

A protonema with a giant nucleus has already been reported forBryopsis hypnoides (Neumann 1969a). The same has now also been found in cultures ofBryopsis monoica. The single nucleus of the protonema reaches about eight times the diameter of the vegetative nuclei in the multinucleate thallus. This giant nucleus contains a long sinuous nucleus similar to that ofAcetabularia mediterranea. Prior to the adventitious formation of the pinnate thalli, transition to the multinucleate stage takes place. The division of the giant nucleus had been observed before inBryopsis hypnoides. The protonema ofBryopsis monoica is capable of holocarp formation of gametes immediately after transition to the multinucleate stage. A mononucleate protonema has further been found inPseudobryopsis (=Trichosolen)myura which fully developed in culture. The protonema is lobed, wound, and irregularly branched. The nucleus increases too, but does not reach the proportions of that ofBryopsis species, and the nucleolus does not become elongate. The transition to the multinucleate stage is accomplished when the development of the pinnate thalli begins. Prior to the formation of the pinnules the young thalli ofBryopsis hypnoides andPseudobryopsis myura resemble each other, especially at their base on the protonema. These similarities, revealed in cultivated specimens, point to a closer taxonomic relation between the generaBryopsis andPseudobryopsis than has hitherto been assumed.     

INTRASPECIFIC INTERACTIONS AND PARASITISM IN AN ASSOCIATION OFRHIZOCARPON LECANORINUMandR. GEOGRAPHICUM

Intraspecific thallus interactions in mixed populations ofRhizocarpon lecanorinumR. geographicumin North Wales are described. In contrast to the most commonly occurring morphotype ofR. geographicumwhich forms mosaics comprised of sharply delimited individual thalli,R. lecanorinumthalli consistently merge with one another at intraspecific contacts, leaving no visible trace of their initial boundaries. On the analogy of mycelial interactions in populations of wood-rotting and other non-lichen-forming fungi, it is postulated that these differences are the outcome, inR. geographicumof somatic incompatibility between distinct genotypes, and inR. lecanorinumof somatic compatibility among genetically identical or very closely related thalli. Possible mechanisms for the evolution of clonal population structure inR. lecanorinumand links between somatic interactions and reproductive output in these species are discussed. Facultative parasitism ofR. lecanorinumbySchaereria fuscocinereaand resistance to such parasitism by co-occurringR. geographicumare also documented.     

Mitochondrial and nuclear DNA diversity within six species ofPhytophthora

The extent of intraspecific mitochondrial DNA (mtDNA) diversity was investigated in isolates ofPhytophthora capsici,P. citricola, P. citrophthora, P. megakarya, P. palmivora, andP. parasitica that represented a wide range of host plants and geographic origins. Phenograms were constructed following the analysis of restriction fragment patterns that were generated by several endonucleases. The six species showed different degrees of mtDNA diversity. Restriction fragment patterns inP. palmivora andP. parasitica were very uniform. Distinct subgroups could be distinguished inP. megakarya andP. citrophthora that correlated with the geographic origin or the host plant, respectively. These subgroups did not seem to be closely related to each other. High degrees of diversity were also evident inP. citricola andP. capsici. Although some isolates ofP. capsici had identical mtDNA patterns, no distinct subgroups were found that could be correlated with either a specific host plant or geographic origin. InP. capsici andP. parasitica variation in nuclear DNA was much more pronounced as compared to mtDNA. InP. capsici both types of analysis correlated well. Because of very limited variation of mtDNA inP. parasitica a comparison between the two phenograms was difficult.     

Heterochromatin (C-bands) in somatic and male germ cells in three species ofMicrotinae

The C-banding patterns in the chromosomes ofMicrotus oeconomus, M. arvalis andM. ochrogaster demonstrate differences in the amount and distribution of heterochromatin. Autosomal centromeric heterochromatin appears as conspicuous blocks or as small dots, and in several chromosomes no heterochromatin was detected; interstitial heterochromatin was observed in one autosome pair ofM. ochrogaster. The sex chromosomes also demonstrate differences in the C-banding pattern. InM. oeconomus, the X chromosome exhibits a block of centromeric heterochromatin which is larger than that of the autosomes; this characteristic helps to recognize the X chromosomes in the karyotype. InM. arvalis no heterochromatin was appreciated in the sex chromosomes. The Y chromosomes ofM. ochrogaster andM. oeconomus are entirely heterochromatic. During male meiosis heterochromatin shows condensation, association and chiasma prevention; the sex chromosomes pair end to end in the three species. At pairing, the Y chromosome ofM. arvalis is despiralized, but it appears condensed again shortly before separation of the bivalent.     

Seasonal acclimation in the epiphytic lichen Parmelia sulcata is influenced by change in photobiont population density

CO₂ gas exchange, radial growth, chlorophyll (Chl) content and photobiont density of an epiphytic population of Parmelia sulcata were monitored every 2 months during 1 year in a temperate deciduous forest of Central Italy, to verify possible seasonal variations. Light response curves of south-exposed thalli, built up in the laboratory at 6 and 27°C at optimal thallus hydration, showed that CO₂ gas exchange changed significantly during the year, with a maximum for gross photosynthesis in December at both temperatures. Photoinhibition phenomena occurred in early spring, immediately before tree leaves sprouted. The principal component analysis of CO₂ gas exchange parameters clearly separated the months with from the months without tree canopy cover. Radial growth, measured on marginal lobes of north- and south-exposed thalli, was the highest in December, and the lowest in April. Photobiont density, measured in lobes of south- and north-exposed thalli with a sedimentation chamber, also changed during the year: the number of photobionts was highest in June and December, and lowest in April, although no significant change in cell size and Chl content per cell was evident throughout the year. South-exposed thalli had slightly, but constantly higher photobiont density both on a weight and an area basis. The acclimation of lichen photosynthesis and Chl content to seasonal temperature and light changes should partially be re-visited on the basis of the significant variation in photobiont population density. This phenomenon still awaits, however, a satisfactory explanation, although it is probably related to the seasonal change in nutrient availability.     

Role of auxin and gibberellin in the synthesis of Ascorbic Acid and growth of tissue explants

Coleoptile and root tips ofTriticum aestivum cv. Arnej 624 and those ofAvena sativa cv. Victory (Svalöf) as well as dry excised embryos ofTriticum aestivum cv. Rival (Svalöf) and those ofArachis hypogaea cv. 34 3A. H. were cultivated in media containing various concentrations of sucrose and growth regulators, like ascorbic acid, indole-3-acetic acid and gibberellin. Growth, differentiation and water uptake of the various explants were determined at regular time intervals. Further, the concentration of the endogenous ascorbic acid in mg./g. fresh weight, as well as the amount of this growth regulator utilized as per cent of the total were determined. Although all the three growth regulators promote growth in the explants, their effect is best felt when sucrose of a higher concentration (1.0 per cent) is added to the medium. In fact, the response to 1.0 per cent sucrose is sometimes as good as a combination of a growth regulator with sucrose, especially in the case of root explants. The results clearly indicate that the biosynthesis of ascorbic acid in the explants is catalyzed by the addition of indole-3-acetic acid as well as gibberellin. Simultaneously, the utilization of ascorbic acid is also appreciably increased by the presence of these growth regulators. Addition of 1.0 per cent sucrose to the medium containing the above mentioned growth regulators augments to a considerable extent not only the concentration of ascorbic acid, but also steps up its utilization. Enhancement of ascorbic acid as well as its increased utilization are correlated with rapid imbibition of water, growth and differentiation. The role of ascorbic acid in growth is discussed; and on the basis of the data presented here it is postulated that: (1) auxin and gibberellin function in the growth process by catalyzing the biosynthesis of ascorbic acid; and (2) that ascorbic acid not only participates in activation of various enzyme systems, but also stimulates the production of adenosine triphosphate by acting as an electron donor in photosynthetic phosphorylation as well as oxidative phosphorylation; (3) that the above action of ascorbic acid creates a favourable redox balance for synthesis of nucleic acids, proteins, enzymeproteins, and cell-wall constituents, thus enabling the processes of cell division and enlargement to proceed at a fast rate; and (4) that the relative rates of cell division and cell enlargement as well as “ageing” will determine the pattern of plant development.     

Concerning the presence and formation of ascorbic acid in yeasts

The selective, sensitive method of analysis of ascorbic acid by high performance liquid chromatography with electrochemical detection (HPLC/EC) has been used to determine the ascorbic acid content of cell extracts from yeasts grown in glucose-free medium, 0.3 M D-glucose, and 0.112 M L-galactono-1,4-lactone. Saccharomyces cerevisiae (strain G-25 and its tetraploid) and a commercial baker's yeast contained less than 2 μg ascorbic acid g^?1 wet wt. of cells when grown for 22 h in glucose-free medium. In 0.3 M D-glucose, only the commercial baker's yeast gave a slight increase (2–50 μg g^?1 wet wt. in 22 h). In 0.112 M L-galactono-1,4-lactone, all three strains produced ascorbic acid (372–587 μg g^?1 wet wt. in 22 h). Lypomyces starkeyi, a species previously reported to contain a significant amount of ascorbic acid (Heick et al., Can. J. Biochem., 47 (1972) 752), was essentially devoid of ascorbic acid under all three conditions of incubation although it did contain an HPLC/EC reactive peak (R_T = 0.87 relative to ascorbic acid) that was readily oxidized by charcoal in the presence of oxygen. The identity of this new compound remains to be determined.     

COMPARATIVE STUDIES ONXANTHORIA PARIETINA,A POLLUTION-RESISTANT LICHEN,ANDRAMALINA DURIAEI,A SENSITIVE SPECIES. I. EFFECTS OF AIR POLLUTION ON PHYSIOLOGICAL PROCESSES

Xanthoria parietinathalli were collected from a ‘ clean-air ’ location and from a polluted area.Ramalina duriaeithalli were collected from the same ‘ clean-air ’ location and some thalli were transplanted to air-polluted locations, whereR. duriaeino longer occurs. The effects of air contaminants on these two lichens were compared under controlled laboratory conditions and in field experiments. Air contaminants and exposure to bisulphite ions had little or no damaging effect onX. parietina, whereas severe damage was caused toR. duriaei, as judged by chlorophyll degradation, autofluorescence of photobionts, photosynthetic activity, membrane integrity and ATP content. The different responses presented confirm the sensitivity ofR. duriaeiand resistance ofX. parietinato air pollution.     

THE IDENTITY OF PHOTOBIONTS FROM THE LICHENPYRENULA JAPONICA

Nine specimens ofPyrenula japonicaKurokawa were collected from various areas in Japan. Photobionts were isolated from their thalli and studied taxonomically. As a result, only one species of green alga,Trentepohlia lagenifera(Hild.) Wille was identified as the photobiont ofPyrenula japonica. This is the first record of this alga from the lichen genusPyrenula.     

Factors influencing the dispersal of a native parasitoid, <Emphasis Type="Italic">Phasgonophora sulcata</Emphasis>, attacking the emerald ash borer: implications for biological control

High parasitism by a native parasitoid, Phasgonophora sulcata Westwood (Hymenoptera: Chalcididae), has been reported on emerald ash borer (hereafter EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), in North America. Use of this parasitoid in an augmentative biological control program has been proposed to slow the spread of EAB, yet information is lacking on key aspects of this parasitoid’s dispersal. We document the flight capacity and walking activity of P. sulcata, its potential fecundity, and describe how age, body size, temperature, and time of day affect these parameters. Wasp flight capacity, measured using flight mills, increased with temperature and decreased with age. Unexpectedly, age and body size did not affect wasp walking activity, and we saw no relationship between walking activity and flight capacity. Older wasps had lower potential fecundity than younger wasps. These results suggest that P. sulcata should be released as pupae near EAB-infested ash trees to improve efficacy and potential biological control success.     

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological,molecular and ecophysiological data

The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20–25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.