Resuscitation and morphological alterations of <Emphasis Type="Italic">Salmonella bovismorbificans</Emphasis> cells under starvation in soil

Salmonella bovismorbificans, rare serovar, isolated from patient in Tunisia were incubated during 13 years in soil. After its resuscitation, the cells showed a biochemical profile completely inactive on Api 20E system. These cells recuperated their initial characters after 6 months of incubation in Tryptic Soy broth. The atomic force micrographs showed a reduction of the cells size and an evolution to coccoid-shapes. After resuscitation S. bovismorbificans cells recuperated their original rod shape. These cells showed an altered envelope. The resuscitate cells were identified by PCR following the amplification of the internal transcribed spacer (ITS) region of 16S–23S rRNA gene.     

Expression and purification of two major outer membrane proteins from <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Vibrio alginolyticus, a Gram-negative bacterium, is one of Vibrio pathogens common to human and marine animals. Outer membrane proteins of bacteria play an important role during infection and induction of host immune response. In present research, two outer membrane protein genes (OmpK and OmpW) of V. alginolyticus were cloned and expressed. The open reading frames of OmpK and OmpW contain 846 bp and 645 bp, respectively, the mature proteins consist of 261 and 193 amino acid residues. At the signal peptides positions −3 to −1, the amino acids were V-M-A in OmpK and V-F-A in OmpW, which consistented with the observed sequence V-X-A of the signal peptides of transmembrane OMP. The alignment analysis indicated that both proteins were highly conserved, which could serve as surface antigens for vaccine candidates. SDS-PAGE indicated two genes over-expressed in E. coli BL21 (DE3). By affinity chromatography on Ni²⁺-nitriloaceate resin, the recombinant proteins were purified from inclusion bodies. Western blot analysis revealed that both proteins had immunoreactivity, which provided a base for further study on the evaluation of diagnostication and vaccine candidates.     

Numerical Taxonomy of <Emphasis Type="Italic">Vibrionaceae</Emphasis> Isolated from Cultured Amberjack (<Emphasis Type="Italic">Seriola dumerili</Emphasis>) and Surrounding Water

A numerical taxonomic study was performed on 148 isolates of Gram-negative, heterotrophic, facultative anaerobic bacteria isolated from amberjack (Seriola dumerili) and its surrounding culture water. The study included 30 type and reference strains belonging to genera Vibrio, Listonella, and Photobacterium. The strains were characterized by 109 morphological, biochemical, physiological, and nutritional tests. Cluster analysis of similarity matrices obtained with S_SM and S_J coefficients was carried out. UPGMA (unweighted pair group mathematical average) analysis defined 11 phena at S_SM values ≥ 86%. Nine phena were identified as Vibrio alginolyticus, V. fischeri, V. harveyi, V. carchariae, V. mediterranei, V. splendidus, V. furnissii, V. parahaemolyticus, and Photobacterium damselae subsp. damselae. The two latter comprised strains isolated from diseased fish. Received: 27 March 2002 / Accepted: 24 May 2002     

Occurrence of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> in the German Bight over a seasonal cycle

Bacteria of the genus Vibrio are an important component of marine ecosystems worldwide. The genus harbors several human pathogens, for instance the species Vibrio parahaemolyticus, a main cause for foodborne gastroenteritis in Asia and the USA. Pathogenic V. parahaemolyticus strains emerged also in Europe, but little is known about the abundance, pathogenicity and ecology of V. parahaemolyticus especially in Northern European waters. This study focuses on V. parahaemolyticus and its close relative Vibrio alginolyticus in the North Sea (Helgoland Roads, Germany). Free-living, plankton-attached and shellfish-associated Vibrio spp. were quantified between May 2008 and January 2010. CFUs up to 4.3 × 10³ N l⁻¹ and MPNs up to 240 N g⁻¹ were determined. Phylogenetic classification based on rpoB gene sequencing revealed V. alginolyticus as the dominant Vibrio species at Helgoland Roads, followed by V. parahaemolyticus. We investigated the intraspecific diversity of V. parahaemolyticus and V. alginolyticus using ERIC-PCR. The fingerprinting disclosed three distinct groups at Helgoland Roads, representing V. parahaemolyticus, V. alginolyticus and one group in between. The species V. parahaemolyticus occurred mainly in summer months. None of the strains carried the virulence-associated genes tdh or trh. We further analyzed the influence of nutrients, secchi depth, temperature, salinity, chlorophyll a and phytoplankton on the abundance of Vibrio spp. and the population structure of V. parahaemolyticus. Spearman Rank analysis revealed that particularly temperature correlated significantly with Vibrio spp. numbers. Based on multivariate statistical analyses we report that the V. parahaemolyticus population was structured by a complex combination of environmental parameters. To further investigate these influences is the key to understanding the dynamics of Vibrio spp. in temperate European waters, where this microbial group and especially the pathogenic species, are likely to gain in importance.     

Rhizosphere competent <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in the management of <Emphasis Type="Italic">Heterodera cajani</Emphasis> on sesame

Biological control of the cyst forming nematode Heterodera cajani was studied on sesame using plant growth promoting rhizobacteria (PGPR) Pseudomonas aeruginosa LPT3 and LPT5. Based on plant growth promoting attributes, two fluorescent pseudomonads, LPT3 and LPT5 were evaluated for their efficacy against cyst forming nematode Heterodera cajani that parasitize Sesamum indicum. Pseudomonas aeruginosa LPT5 produced IAA, HCN, chitinase, glucanase and siderophore, and also solubilized inorganic phosphate in vitro. Moreover, LPT5 resulted in mortality of second stage juveniles of H. cajani, which was 13% higher as compared to P. aeruginosa LPT3. Interestingly, when both strains were inoculated together for the management of H. cajani on Sesamum indicum the population of H. cajani was reduced significantly, in field trial. Approximately 60% reduction in cyst and juveniles population was recorded with LPT5 coated seeds, while LPT3 resulted in 49% reduction in cyst and juvenile population as compared to control. Plants grown with seeds bacterized with LPT5 and reduced doses of urea, diammonium phosphate (DAP), muriate of potash (K) and gypsum gave maximum increase in yield, in comparison to that of plants raised under the influence of recommended or full doses of the chemical fertilizers. Pseudomonas aeruginosa LPT5 also showed excellent root colonization.     

Study on the scavenging and anti-<Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">aureus</Emphasis> activities of the extracts,fractions and subfractions of two <Emphasis Type="Italic">Volvariella volvacea</Emphasis> strains

Scavenging and anti-Staphylococcus aureus activities of extracts, fractions and subfractions from the in vitro mycelium of two strains of the edible mushroom Volvariella volvacea were determined. Chloroform subfractions of the ATCC62890 strain showed the highest inhibition percentage of the DPPH radical (over 80%) after 180 min. When chloroform and hexane subfractions of the R83 strain were combined they showed moderate (inhibition zone of 8.99 ± 0.78 mm) to high (inhibition zone of 13.06 ± 0.41 mm) activity against the Gram-positive bacteria Staphylococcus aureus, which are 74.4 and 51.2% of tetracycline (inhibition zone of 17.55 ± 0.11 mm). Partitioning suggested that the substances in the chloroform and hexane fractions of the R83 strain act synergistically to give the antimicrobial activity, while separating the substances of the ATCC62890 subfractions reduced their activity. In general, the R83 strain seems to be a source of antimicrobial substances, while the ATCC62890 strain appears to be an alternative source of antioxidants.     

Geometric characters of the radius and tibia in <Emphasis Type="Italic">Macaca mulatta</Emphasis> and <Emphasis Type="Italic">Macaca fascicularis</Emphasis>

We performed comparative analyses of four cross-sections of the distal radius and tibia in two species of macaque to clarify the relationships between bone morphology and locomotor type. The lengths of bones and five bone geometric properties in each section were examined and compared separately in both female and male Macaca mulatta and Macaca fascicularis. In M. mulatta, there were no significant gender-specific differences in either the radius or the tibia. In contrast, the radius and tibia of male M. fascicularis had greater geometric parameters in the 20% and 40% positions relative to the 5% and 10% positions from the distal end than those of their female counterparts. The radius and tibia of M. mulatta were relatively longer than those of M. fascicularis, and the sectional parameters of the tibia of M. mulatta were relatively larger than those of M. fascicularis. Standardization of the log-transformed bone length between the species revealed larger radial cortical bone areas in M. fascicularis. In contrast, there were minimal differences in the tibial cortical bone areas between the two species. This study suggests that the observed distinctions in bone geometry in female and male M. fascicularis may be due to gender-specific differences in the muscle weights of the forearm and calf, which may underlie the divergence in the leaping abilities of females and males of this species. Taken together, these results of interspecies comparisons may be related to the fact that arboreal primates such as M. fascicularis undergo compressive mechanical stress due to the forelimb lead that occurs as the animal descends a sloping trunk or bridges a tree gap downward, while terrestrial primates such as M. mulatta move on nearly flat substrates. Differences in fore- and hind-limb bone properties between the two species are discussed with regard to functional morphology and locomotor type.     

Isolation and characterization of porins from <Emphasis Type="Italic">Desulfovibrio piger</Emphasis> and <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>: structure and gene sequencing

The outer membrane proteins of Desulfovibrio piger and Bilophila wadsworthia (Omp-DP and Omp-BW, respectively) and the genes encoding them (omp-DP and omp-BW) were isolated and characterized. Native Omp-DP and Omp-BW form a trimeric structure of approximately 120 kDa. These proteins disaggregated into monomers with a molecular weight of approximately 53 kDa after heating at 95°C for 10 min. The pore-forming abilities of these oligomeric proteins demonstrated that they form small nonspecific channels with an exclusion limit of 260–300 Da. The omp-DP and omp-BW genes were cloned and sequenced. Sequence analyses revealed an open reading frame of 1,512 bp for omp-DP and 1,440 bp for omp-BW. The mature Omp-DP protein consisted of 480 amino acids and had a calculated MW of 53,290 Da. The mature Omp-BW protein consisted of 456 amino acids and had a calculated MW of 50.050 Da. Alignment of Omp-DP with Omp-BW revealed 54% homology, whereas alignment with other known porins showed a low level of homology. Analysis of the secondary structures indicated that both proteins span the outer membrane 18 times with amphipathic β-strands. This research presents porins which were isolated and characterized for the first time from bacteria belonging to the Desulfovibrionaceae family. O. Avidan and E. Kaltageser have contributed equally to this work.     

<Emphasis Type="Italic">Fopius arisanus</Emphasis> oviposition in four <Emphasis Type="Italic">Anastrepha</Emphasis> fruit fly species of economic importance in Mexico

We studied the oviposition performance of Fopius arisanus (Sonan) (Hymenoptera: Braconidae) attacking eggs of four fruit flies of the genus Anastrepha Schiner (Diptera: Tephritidae) under laboratory conditions. The complete process of oviposition on an individual egg of Anastrepha ludens (Loew) lasts in average 85.4 ± 2.9 s, including a tremor (25.8 ± 1.03 s) observed in the middle of this process related to the egg’s descent. The average parasitism of A. ludens egg was 60.9 ± 7.5%, with only 1.2% of superparasitized eggs. During individual acts of oviposition, we noted that F. arisanus possesses a highly flexible ovipositor that curves easily as it searches for additional suitable eggs, which may be of particular benefit when a female finds large clutches of eggs. The individual oviposition of F. arisanus in host fruits attacked by Anastrepha spp. varies with the egg clutch size of each fruit fly species: A. serpentina laid the biggest egg clutches (21.3 ± 1.4), followed by A. ludens (14.2 ± 0.9), and A. striata (1.0 ± 0.0) (=A. obliqua). The time spent by F. arisanus in individual ovipositions was parallel to these findings, reinforcing the idea that F. arisanus attacks several eggs in each individual insertion of its ovipositor. Neither formal oviposition acts, nor adult emergences of F. arisanus were registered in A. obliqua. We discuss the potential of F. arisanus as natural enemy of fruit flies of the genus Anastrepha, and explore the eventual developing of its mass rearing. Handling Editor: Torsten Meiners.     

A new name in <Emphasis Type="Italic">Pavetta</Emphasis> L. (<Emphasis Type="Italic">Rubiaceae</Emphasis>)

Summary  The name Pavetta modesta (Hiern) S. E. Dawson is a later homonym of P. modesta Bremek. Pavetta crystalensis is proposed as a new name.     

Photosynthetic and transpiration responses of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated <Emphasis Type="Italic">Solanum nigrum</Emphasis> L. plants to <Emphasis Type="Italic">ex vitro</Emphasis> adaptation

In vitro regeneration of black nightshade (Solanum nigrum L.) plants was achieved through callus-mediated shoot organogenesis followed by 30 d indoor ex vitro adaptation to nutritional stress under environmental ambience and thereafter 6-d outdoor acclimatization in pots prior to field establishment. Relevant physiological parameters including pigment content, chlorophyll a fluorescence, net photosynthetic rate (P _N), transpiration rate (E), and stomatal conductance (g _s) of in vitro-regenerated plants were investigated during the course of ex vitro adaptation. During the first 4 d of indoor transplantation to potting substrate, there was a marginal reduction in the leaf chlorophyll and carotenoid contents but P _N and E were strongly reduced. The stomatal conductance and E/P _N ratio were significantly higher in plants up to 20 d of indoor adaptation than those of comparable age grown naturally from seeds. The shape of the OJIP fluorescence transient varied significantly with acclimatization, and the maximum change was observed at 2.0 ms. The 2.0 ms variable fluorescence (V _j), 30 ms relative fluorescence (M ₀), photon trapping probability (TR₀/Abs), and photosystem II (PSII) trapping rate (TR₀/RC) showed initial disturbance and subsequent stabilization during 30 d of indoor acclimatization. Energy dissipation (DI₀/RC) and electron transport probability (ET₀/TR₀) showed an initial phase of increase during the 4 d after plants were transplanted outdoors. During the 6-d outdoor acclimatization after transfer of plants to soil, no significant change in total chlorophylls and carotenoids, E, and g _s were observed, but P _N improved after reduction on the first d. The OJIP-derived parameters experienced change on the first d but were stabilized quickly thereafter. There was no significant difference between outdoor acclimatized plants and those of the seed-grown plants of comparable age with respect to photosynthetic and fluorescence parameters. Direct transfer of plants without indoor acclimatization, however, showed a completely different trend with respect to P _N, E, and OJIP fluorescence transients. The bearing of this study on optimizing micropropagation is discussed.     

Genotype Analysis of Collagenase Gene by PCR-SSCP in <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> and its Association with Virulence to Marine Fish

In this article, the genotypes for Vibrio alginolyticus were analyzed based on collagenase gene by PCR-single strand conformation polymorphism (SSCP) method and their relationship to the virulence for marine fish were studied by a bacteria challenging experiment. Results showed total 13 genotypes designated as A1, A2, A3, A4, B, C, D, E, F, G1, G2, H, and I were identified. Among them, isolates from type A1 were dominant in abundance (48.1%) and the isolates from Nanyou had the highest polymorphisms, in which 11 genotypes were identified from 25 isolates. Moreover, the bacteria challenging experiment showed the virulence of V. alginolyticus was correlated to that of the collagenase-SSCP type (r = 0.438, P < 0.01), which demonstrated the strains from type A1, A2, A3, B, C, F, G1, and I were low-virulent isolates, while those from type A4, D, E, G2, and H were high virulence. These data will provide a new understanding of the relationship between virulence of V. alginolyticus and its molecular marker.     

<Emphasis Type="Italic">Pseudocercospora griseola</Emphasis> Causing Angular Leaf Spot on <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> Produces 1,8-Dihydroxynaphthalene-Melanin

Pseudocercospora griseola is the causal agent of angular leaf spot of common bean (ALS). It has undergone parallel coevolution with its host and two major groups have been defined, “Andean” (P. griseola f. griseola) and “Mesoamerican” (P. griseola f. mesoamericana). The aim of this study was to analyze the nature and the level of the dark pigment synthesized by the representatives of each group. After 21 days of incubation on potato dextrose agar medium, P. griseola f. griseola isolate S3b developed colonies with diameters of 17.5 ± 1.3 mm and concentric rings of pigmentation. Isolate T4 of P. griseola f. mesoamericana presented smaller colonies (9.9 ± 0.3 mm) with a uniform dark-gray color. Both isolates, S3b and T4, produced the same pigment, a 1,8-dihydroxynaphthalene-melanin, although different in quantity and structural features as suggested by the IR spectrum. The P. griseola f. griseola isolate S3b had a higher growth rate and melanin content as well as smaller sensitivity to melanin synthesis inhibitors compared to the isolate T4 of P. griseola f. mesoamericana. These results suggest a possible link between melanin and growth in P. griseola.     

Growth and carrageenan quality of <Emphasis Type="Italic">Kappaphycus striatum</Emphasis> var. <Emphasis Type="Italic">sacol</Emphasis> grown at different stocking densities,duration of culture and depth

Kappaphycus striatum var. sacol was grown in two separate studies: (1) at two stocking densities, and (2) at four different depths, each for three different durations of culture (30, 45 and 60 days) in order to determine the growth rate of the seaweed and evaluate the carrageenan content and its molecular weight. The results demonstrated that stocking density, duration of culture and depth significantly (P < 0.01) affected the growth rate, carrageenan content and molecular weight of K. striatum var. sacol. Decreasing growth rate was observed at both stocking densities and at four depths as duration of culture increased. A lower stocking density (500 g m⁻¹line⁻¹) showed a higher growth rate for the shortest durations, i.e. 30 days, as compared to those grown at a higher density. Likewise, decreasing growth rate was observed as depth increased, except at 50 cm after 60 days of culture. A 45-day culture period produced the highest molecular weight at both stocking densities (500 g m⁻¹line⁻¹ = 1,079.5 ± 31.8 kDa, 1,000 g m⁻¹line⁻¹ = 1,167 ± 270.6 kDa). ‘Sacol’ grown for 30 days at 50 cm (1,178 kDa) to 100 cm (1,200 kDa) depth showed the highest values of molecular weight of carrageenan extracted. The results suggested that K. striatum var. sacol is best grown at a stocking density of 500 g m⁻¹line⁻¹, at a depth of 50–100 cm, and for a duration of 30 days in order to provide the highest growth rate, carrageenan content and molecular weight.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Evaluation of the V404I and V509M amino acid substitutions of <Emphasis Type="Italic">ERG11</Emphasis> gene in <Emphasis Type="Italic">Candida albicans</Emphasis> isolates by pyrosequencing

The molecular mechanisms underlying fluconazole resistance in C. albicans involve mutations and the overexpression of the ERG11 gene and membrane transport proteins. We examined the relationship between the reduced fluconazole susceptibility of C. albicans and mutations of V404I and V509M in the ERG11 gene in 182 C. albicans clinical isolates using the Pyrosequencing™ method. DNAs from these clinical isolates with different levels of in-vitro fluconazole susceptibility — one resistant, five susceptible dose-dependent (SDD), four trailer, and 172 susceptible — were analyzed. None of the fluconazole-susceptible, SDD, trailer or resistant isolates had mutations of V404I or V509M. Our results showed that no correlation can be found between the V404I or V509M mutation and fluconazole susceptibility in C. albicans.