Site-specific integration of the phage ΦCTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP

The site-specific integration of the phage ?CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage ?CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.     

The potential of Beauveria bassiana and Metarhizium anisopliae in controlling the root-knot nematode Meloidogyne incognita in tomato and cucumber

Meloidogyne incognita is one of the most important plant parasitic nematodes. This study was conducted to determine the nematicidal potential of Beauveria bassiana and Metarhizium anisopliae against M. incognita. B. bassiana and M. anisopliae spore suspensions and bio -nematicide, Purpureocillium lilacinum were applied. B. bassiana and M. anisopliae revealed considerable nematicidal activity against M. incognita in tomato and cucumber. The gall index decreased gradually from 8.0 for control to 3.2, 2.0 and 2.2 for B. bassiana, M. anisopliae and P. lilacinum in tomato, respectively. The highest reduction (%) in gall formation (control index) was calculated as 75.2 % in M. anisopliae treated group for tomato. The gall index was 7.6 for control, but decreased to 3.6, 2.0 and 2.2 for B. bassiana, M. anisopliae and P. lilacinum in cucumber, respectively. The highest control index was 71.7 % for M. anisopliae in cucumber. The number of the second instar juveniles of M. incognita was recorded as 2240 for control. However, this value reduced to 508, 332 and 328 by B. bassiana, M. anisopliae and P. lilacinum in tomato, respectively. The highest control indexes for the second instar juveniles were 85.2 % and 85.3 % for M. anisopliae and P. lilacinum in tomato, respectively. Alike, the highest control indexes were 84.9 % and 85.7 % for the same applications in cucumber, respectively. B. bassiana and M. anisopliae displayed also positive effect on the number of leaves, dry and fresh root weights of tomato. The results showed that B. bassiana and M. anisopliae can be considered as an alternative.     

Occurrence of pathogens in Ips typographus (Coleoptera: Curculionidae) and in other spruce bark beetles from the wilderness reserve Dürrenstein (Lower Austria)

The study presents new data on spatial distribution of bark beetle pathogens, on changes in frequency over several years and on their prevalence during different time periods within a year from several locations within the wilderness reserve Dürrenstein (Lower Austria). The occurrence of pathogens was investigated in Ips typographus (during five years), in Pityogenes chalcographus (during two years) and in Ips amitinus (in one year). In total, seven pathogen species could be detected in I. typographus. The most dominant pathogen species were the Ips typographus-Entomopoxvirus (ItEPV), the sporozoan species Gregarina typographi and the microsporidium Chytridiopsis typographi; the latter two pathogen species were recorded every year and at about similar high (G. typographi) or low (C. typographi) rates, the ItEPV in strongly varying rates. The neogregarine Mattesia cf. schwenkei and the two microsporidia Nosema typographi and Unikaryon montanum were found in I. typographus only sporadically and the rhizopodan species Malamoeba scolyti was found once. The number of infected males and females was relatively similar with almost all pathogen species in most of the years except U. montanum, which occurred exclusively in females. Three pathogen species were recorded in P. chalcographus which were Gregarina typographi, Mattesia cf. schwenkei and Chytridiopsis typographi. Two pathogen species were observed in I. amitinus, Gregarina typographi and Chytridiopsis typographi.     

Evaluation of koji prepared with various molds for mirin-making

Koji is one of the raw materials used for mirin-making, and it is traditionally prepared with Aspergillus oryzae. To improve productivity and to be available for a variety of mirin, various koji were prepared with A. niger, A. awamori, A. usamii mut. shiro-usamii, and Rhizopus oligosporus and examined for possible to use in mirin-making. The degrees of digestion of the starch and protein in various koji were at its highest with either koji prepared with A. oryzae at pH 7.0 or with A. usamii mut. shiro-usamii at pH 3.0. The degree of digestion of the protein in koji made with A. usamii mut. shiro-usamii was decreased in koji digestion at higher temperature (at over 50°C) compared to that in koji made with A. oryzae. We examined the ratio of koji to steamed glutinous rice to identify the optimum value for mirin production. The optimum ratios for digestion of starch in the mash with koji of A. oryzae and A. usamii mut. shiro-usamii were 0.10 and 0.15, respectively. These values were in agreement with the 0.10–0.25 established by experience. Therefore, of the five strains tested, A. usamii mut. shiro-usamii, among the tested molds was the most suitable strain for preparing koji in mirin-making without decreasing productivity, while lending new qualities to the mirin. The koji prepared with A. oryzae had a good smell but the koji prepared with A. usamii mut. shiro-usamii had a little mold smell. Then if this little mold smell could be improved, this koji can be used in mirin-making. The koji prepared with A. niger contained the most citric acid of all strains but the digestivities of starch and protein in the koji was less than those of koji prepared with A. oryzae and A. usamii mut. shiro-usamii. The digestivities of starch and protein in koji prepared with A. awamori were the lowest values. In 35% alcohol, its productivities in mirin-making seemed to be low. Because of the highest content of lactic acid, the koji prepared with R. oligosporus seemed to be suitable for mirin-making. Then if its productivity could be improved, this koji can be used in mirin-making.     

Intertidal zonation of prosobranch gastropods: Analysis of densities of four co-existing species

The densities of populations of Nerita atramentosa Reeve, Austrocochlea constricta Lamarck, Bembiciuin nanum (Lamarck), and Cellana tramoserica (Sowerby) were recorded in 0.25 m² quadrats on landward and seaward halves of a sheltered and a moderately exposed rock platform from June 1972 to June 1973. The data have been subjected to variance and multiple regression analysis. Differences between densities of each species on the two shores and in the two areas of each shore are discussed with respect to the regressions on littoral height. Densities of Nerita on the sheltered shore, Austrocochlea and Bembicium on both shores, and Cellana, on the exposed shore, increased with height. The density of Cellana on the sheltered shore decreased as height increased. The density differences in the landward and seaward areas on the two shores were attributable to the effect of height for Nerita and Austrocochlea. Height was the major variable affecting the density of these two species on the sheltered shore. Bembicium and Cellana densities on both shores, and Austrocochlea density on the exposed shore, are not primarily determined by height.On the sheltered shore, Nerita and Cellana increased in density with increasing cover of water in rock pools. On the exposed shore, Bembicium and Cellana became sparser with increasing water cover. Bembicium on both shores increased in density with increasing cover of the alga Peyssonelia, as did Cellana on the exposed shore. Cellana and Austrocochlea tended to increase in density with Nerita on the sheltered shore. Cellana tended to be sparse where Bembicium was dense on the exposed shore. The biological effects of substrata and other species on the densities of each species remain unexplained.Significant time effects were found for variations in the densities of Nerita and Cellana on the sheltered shore and of Austrocochlea and Cellana on the exposed shore. The seasonal change in density of Nerita was parabolic with a minimum in summer, and is consistent with the known period of recruitment of this species. The lack of significant time effects on density of Austrocochlea on the sheltered shore is consistent with the known continuous recruitment of this species. The declining density of Austrocochlea on the moderately exposed shore was found, by size-frequency analysis, to be due to reduced recruitment compared with surrounding shores. The density of Cellana on both shores showed seasonal changes, with a maximum in summer, which is consistent with the known period of recruitment in Cellana. The lack of seasonal changes in density of Bembicium is briefly discussed.This type of multifactor analysis identifies biological interactions affecting variation in density of these species. It also corroborates the results of other ecological investigations and determines the relative importance of a variety of physical and biological factors in the distribution of density of intertidal gastropods.     

Taxonomic update of Clitocybula sensu lato with a new generic classification

The taxonomy and phylogeny of the hydropoid clade (genera Clitocybula s.l., Megacollybia, Leucoinocybe gen. nov., Hydropus, Trogia, Gerronema, Porotheleum and Lignomphalia gen. nov.) in Europe is studied using morphological and molecular approaches; the first three genera in detail including all known European species. Only two European species remain in Clitocybula s.str., Clitocybula lacerata and Clitocybula familia. The European C. lacerata is a species complex which should be treated as C. lacerata agg. at the current state of knowledge. A neotype originating from type area was designated to fix the application of the name. The presence of American species Clitocybula abundans in Europe is insufficiently proved. “Clitocybula dryadicola ? belongs to the genus Hydropus, and Clitocybula tilieti has an unclear systematic position. The results showed that Megacollybia and Leucoinocybe represent independent genera separated from Clitocybula. The genus Leucoinocybe is validly published with two European species, Leucoinocybe lenta and Leucoinocybe taniae. “Clitocybula flavoaurantia” proved to be conspecific with the latter species. The genus Lignomphalia is published for “Pseudoomphalina lignicola”, a lignicolous omphalinoid species. The Indian “Clitocybula sulcata” is transferred to Leucoinocybe and “Clitocybula atrialba” to Gerronema. The first European records of Megacollybia marginata are published.     

The rate of allelism of lethal genes in a geographically structured population

The expected rate of allelism, E[I(x)], of lethal genes between two colonies with distance x in a structured population is studied by using one- and two-dimensional stepping-stone models. It is shown that E[I(x)] depends on the magnitude of selection in heterozygous condition (h), the rate of migration among adjacent colonies (m), the number of loci which produce lethal mutations (n) and the effective population size of each colony (N).——E[I(x)] always decreases with distance x. The rate of decrease is affected strongly by the magnitude of m. The rate of decrease is faster when m is small. E[I(x)] also decreases with increasing N and n. The effect of h on E[I(x)] is somewhat complicated. However, E[I(0)] is always smaller when h is small than when it is large.——For large x, the following approximate formulae may be obtained: (see PDF) where q and Var (q) are the mean and the variance of gene frequencies in each colony, t is approximated as t=h, (see PDF), -h for the partially recessive, completely recessive, and overdominant lethals, respectively, and C₀ is a function of m and t. It is clear that E[I(x)] declines exponentially with x in a one-dimensional habitat. The decrease E[I(x)] is faster in a two-dimensional habitat than in a one-dimensional habitat. The present result is applied to some of the existing data and the estimation of population parameters is also discussed.     

Effects of methylbenzenes on microsomal enzymes in rat liver,kidney and lung

The effects of pretreatment with toluene, o-, m-, p-xylene and mesitylene were investigated on the microsomal enzymes of liver, kidney and lung in rats. The activities of aminopyrine N-demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase, NADPH-cytochrome c reductase, as well as the concentrations of cytochrome P-450 and cytochrome b₅ were determined. The effects were most marked in the liver, where toluene caused increase in aniline hydroxylase and cytochrome P-450; o-xylene in aminopyrine N-demethylase and cytochrome b₅; m-xylene and mesitylene in all the enzymes investigated. In kidneys, all the compounds increased the activity of aniline hydroxylase; m-xylene induced cytochrome P-450 and b₅ as well as NADPH-cytochrome c reductase; p-xylene induced cytochrome P-450, and mesitylene cytochrome P-450 and b₅. Aminopyrine N-demethylase activity was decreased by toluene. In lungs, only mesitylene caused any significant differences from the controls: increase in aminopyrine N-demethylase and aryl hydrocarbon hydroxylase, decrease in aniline hydroxylase. The methylbenzenes tested induced the microsomal enzymes in a rough correlation to the number of their methyl groups and their hydrophobic properties.     

Genomic structure and sequence of the pufferfish (Fugu rubripes) growth hormone-encoding gene: a comparative analysis of teleost growth hormone genes

A nested polymerase chain reaction (PCR) technique for amplifying a fragment of the gene (GH) encoding teleost growth hormone has been developed. Using this technique, a fragment of the pufferfish, Fugu rubripes and Arothron maculatus; dwarf gourami, Colisa lalia; guppy, Poecilia reticulata; and goldfish, Carassius auratus GH genes were cloned. The Fugu rubripes (Fugu) gene fragment was used to isolate the GH gene from a Fugu genomic library. The complete nucleotide sequence of a 8.5-kb SacI genomic fragment containing the Fugu GH gene has been determined. The GH gene spans 2.5 kb from the first codon to polyadenylation signal, and contains six exons and five introns similar to the GH genes of salmonids, tilapia, barramundi, flounder and yellowtail. The GH introns contain microsatellite and satellite sequences. The microsatellites found in the fifth intron of the GH gene are also present in the corresponding introns of tilapia, barramundi and flounder GH genes. Southern analysis revealed that the GH gene is a single-copy gene in the Fugu. The promoter region of the Fugu GH gene contains conserved sequences that are likely to be involved in the pituitary-specific expression of the gene. A phylogenetic tree of nucleotide (nt) sequences of all known teleost GH genes has been inferred using the distance matrix method. The topology of this tree reflects the major phylogenetic groupings of teleosts. The intron patterns and repetitive sequences of GH genes can serve as useful natural markers for the classification and phylogenetic studies of teleosts.     

Analysis of DNA sequences of six chloroplast and nuclear genes suggests incongruence, introgression, and incomplete lineage sorting in the evolution of Lespedeza (Fabaceae)

The genus Lespedeza (Fabaceae) consists of 40 species disjunctively distributed in East Asia and eastern North America. Phylogenetic relationships of all Lespedeza species and closely related genera were reconstructed using maximum parsimony, maximum likelihood, and Bayesian analyses of sequence data from five chloroplast (rpl16, rpl32-trnL, rps16-trnQ, trnL-F, and trnK/matK) and one nuclear (ITS) DNA regions. All analyses yielded consistent relationships among major lineages. Our results suggested that Campylotropis, Kummerowia, and Lespedeza are monophyletic, respectively. Lespedeza is resolved as sister to Kummerowia and these two together are further sister to Campylotropis. Neither of the two subgenera, subgen. Lespedeza and subgen. Macrolespedeza, in Lespedeza based on morphological characters, is recovered as monophyletic. Within Lespedeza, the North American clade is retrieved as sister to the Asian clade. The nuclear and chloroplast markers showed incongruent phylogenetic signals at shallow-level phylogeny, which may point to either introgression or incomplete lineage sorting in Lespedeza. The divergence times within Lespedeza and among related genera were estimated using Bayesian approach with BEAST. It is assumed that following the divergence between Kummerowia and Lespedeza in Asia in the late Miocene, the ancestor of Lespedeza diverged into the North American and the Asian lineages. The North American ancestor quickly migrated to North America through the Bering land bridge in the late Miocene. The North American and Asian lineages started to diversify almost simultaneously in the late Miocene but resulted in biased numbers of species in two continents.     

Repartition des flavonols dans l'epaisseur des feuilles de quelques vegetaux vasculaires

The tissue localisation of flavonoids has been studied in leaves of Betula, Corylus, Fagus, Fraxinus, Pisum, Platanus, Quercus, Spinacia and Tilia and scales of onion bulbs. All these species contain flavonols which are, for the most part, located in the upper epidermis of the leaves. In the onion bulb, flavonols are exclusively in the epidermis. The flavonols are glycosylated and dissolved in the vacuoles. The leaves were fractionated by an original technique of abrasion of the frozen material. The physiological significance of such a distribution of flavonoids in the adult leaves or scales is discussed.     

Review of the Palaearctic Acomopterella Zaitzev (Diptera,Sciaroidea, Mycetophilidae)

The distribution of Acomopterella species in the Palaearctic region has been re-examined in this study, using recently collected material. The European species was found to be distributed in the eastern Palaearctic as well. A second Palaearctic species from Honshu (Japan) is herein described. The morphology of adult specimens was studied by light microscopy and scanning electron microscopy. The shape of functional specialized setae on mid tibiae in Acomopterella and seven further fungus gnat genera is described and the suitability of this character for systematic studies is discussed. Details of a “hind tibial organ” are described.The position of Acomopterella in the tribe Gnoristini is briefly discussed. Acomopterella is found to be more closely related to Speolepta Edwards, 1925, than to any other recent genus.     

Molecular and morphological evolution in the Physciaceae (Lecanorales,lichenized Ascomycotina), with special emphasis on the genus Rinodina

Abstract:A phylogenetic hypothesis based on nuclear ITS sequence data is presented for the familyPhysciaceae , based on various representatives of foliose and fruticose groups and a number of species selected from the crustose genera Rinodina and Buellia s.l. The analysis supports the monophyly of the Physcia - and theBuellia -groups. This is in agreement with existing morphological evidence, particularly ascus characters. ThePhyscia group in the analysis includes the genera Anaptychia, Heterodermia, Hyperphyscia,Mobergia , Phaeophyscia, Phaeorrhiza, Physcia, Physconia, Rinodina, andRinodinella , while the Buellia group includes Amandinea, Buellia and Diploicia. The genera Physcia, Phaeophyscia, Phaeorrhiza and Rinodinella were well supported as monophyletic groups. The support for Physconia is low. Rinodina and Buellia are not supported as monophyletic genera. In agreement with ascus and ascospore characters, Buellia lindingeri is placed within the Rinodina group, close to R. lecanorina. The genus Amandinea as currently circumscribed was not supported as a monophyletic group. The analysis confirms results from other lichen families that foliose members have evolved more than once from crustose lichens.Rinodina and Rinodinella species without chemical compounds in their thalli form the sister group toPhaeophyscia , and both groups form a monophyletic assemblage. A more detailed analysis of the Physcia group is presented. Whilst several of the foliose genera were well supported, there is only poor support for traditionally accepted crustose genera. The taxonomic implications of these findings are discussed.     

Identification and genetic characterization of the Pseudomonas aeruginosaleuB gene encoding 3-isopropylmalate dehydrogenase

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-β-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway.     

Algicidal effects on Heterosigma akashiwo and Chattonella marina (Raphidophyceae), and toxic effects on natural plankton assemblages by a thiazolidinedione derivative TD49 in a microcosm

The algicidal effects of the thiazolidinedione derivative TD49 on Heterosigma akashiwo and Chattonella marina (Raphidophyceae) were assessed, and the response of the planktonic community and environment to the algicide was evaluated in a microcosm, quantifying 12 L. The abundance of over 80 % of H. akashiwo and C. marina declined in a day significantly in microcosms to which TD49 was added (final concentration 2 μM), and this was correlated with an abrupt decline in the culture pH. The number of protists (i.e., ciliates) other than H. akashiwo and C. marina gradually increased with time in the TD49 treatments, implying that the decline in numbers of H. akashiwo and C. marina cells resulting from TD49 treatment was a major factor in the growth of the other organisms. However, TD49 may be toxic to aquatic zooplankton communities, even though it is a highly selective algicide for harmful algae bloom species. The study indicates that TD49 is an effective agent for the control for H. akashiwo and C. marina blooms in enclosed and eutrophic water bodies.     

New records of Lophoproctus coecus Pocock, 1894 (Diplopoda,Polyxenida, Lophoproctidae) extend the range of the genus Lophoproctus

The geographic distribution of the genus Lophoproctus Pocock, 1894 has greatly expanded with new records of the species Lophoproctus coecus Pocock, 1894, together with the reassignment of a number of millipedes formerly identified as Lophoproctus lucidus (Chalande, 1888). Lophoproctus coecus was found to be the sole representative of the family Lophoproctidae in collections examined from Crimea and the Caucasian region. The species was also identified from Iran and Kyrgyzstan. Lophoproctus specimens collected in Italy by Verhoeff were reassigned as Lophoproctus coecus with the exception of one specimen of Lophoproctus jeanneli (Brölemann, 1910) from Capri. These data were combined with all available information from the literature to look at the pattern of distribution of the four species in the genus. The range of the genus Lophoproctus extends from Portugal to Central Asia. Lophoproctus coecus is widespread from Italy eastward, while the morphologically very similar species Lophoproctus lucidus is confined to France and northern Africa. The two species have a narrow overlap in the Alpes Maritimes region of France. Lophoproctus jeanneli has a scattered coastal distribution around the Mediterranean Sea. The troglobitic species Lophoproctus pagesi (Condé, 1982) has only been recorded from a cave on Majorca, Spain.