Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

Reactive oxygen and nitrogen species’ effect on <Emphasis Type="Italic">lux</Emphasis>-biosensors based on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

The effect of reactive oxygen and nitrogen species on lux-biosensors based on the Escherichia coli K12 MG1655 and Salmonella typhimurium LT2 host strains was investigated. The bioactivity of exogenous free radicals to the constitutively luminescent E. coli strain with plasmid pXen7 decreased in the order H₂O₂ > OCl^– > NO^? > RОO^? > ONOO^–> O₂^?- while the bioluminescence of S. typhimurium strain transformed with this plasmid decreased in the order NO^? > H₂O₂ > ONOO^– > RОO^? > OCl^– > O₂^?- The cross-reactivity of induced lux-biosensors to reactive oxygen and nitrogen species, the threshold sensitivity and the luminescence amplitude dependences from the plasmid specificity and the host strain were indicated. The biosensors with plasmid pSoxS′::lux possessed a wider range of sensitivity, including H₂O₂ and OCl^–, along with O₂^?- and NO^?. Among the used reactive oxygen and nitrogen species, H₂O₂ showed the highest induction activity concerning to the plasmids pKatG′::lux, pSoxS′::lux and pRecA′::lux. The inducible lux-biosensors based on S. typhimurium host strain possessed a higher sensitivity to the reactive oxygen and nitrogen species in comparison with the E. coli lux-biosensors.     

Is there selection by species diversity in <Emphasis Type="Italic">Picea</Emphasis> <Emphasis Type="Italic">abies</Emphasis> L.?

In view of the progressive destruction of the biosphere, biodiversity research plays an important role in the search for factors affecting species diversity and in the analysis of the effects of species diversity on ecosystems. However, research into how species diversity and composition are related to genetic structures is still at the beginning. In this study, we combined population genomics, new correlative approaches, and randomization permutation methods to identify adaptive genetic differentiation in populations of the Norway spruce Picea abies L. that display different levels of species diversity. We detected a significant positive large covariation (C) between species diversity (ν _a ) and genotype frequency at a gene locus, resulting from differential selection by species diversity or by environmental factors. The results may contribute to understand the interplay between ecological and evolutionary processes in determining community structure and dynamics.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

Does sequence polymorphism of <Emphasis Type="Italic">FLC</Emphasis> paralogues underlie flowering time QTL in <Emphasis Type="Italic">Brassica </Emphasis><Emphasis Type="Italic">oleracea</Emphasis>?

Previous locations of flowering time (FT) QTL in several Brassica species, coupled with Arabidopsis synteny, suggest that orthologues of the genes FLC, FY or CONSTANS might be the candidates. We focused on FLC, and cloned paralogous copies in Brassica oleracea, obtained their genomic DNA sequences, and confirmed their locations relative to those of known FT-QTL by genetical mapping. They varied in total length mainly due to the variable size of the first and last introns. A high level of identity was observed among Brassica FLC genes at the amino acid level but non-synonymous differences were present. Comparative analysis of the promoter and intragenic regions of BoFLC paralogues with Arabidopsis FLC revealed extensive differences in overall structure and organisation but showed high conservation within those segments known to be essential in regulating FLC expression. Four B. oleracea FLC copies (BoFLC1, BoFLC3, BoFLC4 and BoFLC5) were located to their respective linkage groups based on allelic sequence variation in lines from a doubled haploid population. All except BoFLC4 were within the confidence intervals of known FT-QTL. Sequence data indicated that relevant non-synonymous polymorphisms were present between parents A12DHd and GDDH33 for BoFLC genes. However, BoFLC alleles segregated independently of FT in backcrosses while the study provided evidence that BoFLC4 and BoFLC5 contain premature stop codons and so could not contribute to flowering time variation. Therefore, there is strong evidence against any of the 4 BoFLC being FT-QTL candidates in this population.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.     

Conjugal transfer using the bacteriophage ϕC31 <Emphasis Type="Italic">att</Emphasis>/<Emphasis Type="Italic">int</Emphasis> system and properties of the <Emphasis Type="Italic">attB</Emphasis> site in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis>

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage ϕC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl₂ without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the ϕC31 att/int system.     

Effect of cold stratification on seed germination in <Emphasis Type="Italic">Solidago</Emphasis> × <Emphasis Type="Italic">niederederi</Emphasis> (<Emphasis Type="Italic">Asteraceae</Emphasis>) and its parental species

In this study, we investigated the influence of cold stratification on seed germination in S. × niederederi, a hybrid between the North American S. canadensis and the European S. virgaurea, using fruit samples collected in 2016 in Poland. We aimed to test the hypothesis that the low temperature exposure decreases the final percentage and speed of seed germination in the hybrid and its parental species. For each species, sets of 100 achenes in three replications were mixed with dry sand and stored in Petri dishes in darkness for 12 weeks, at ?18 °C and?+?4 °C, and?+?25 °C. The seeds were incubated for 21 d at room temperature (+25 °C), under the 12 h photoperiod (630 lx). We showed a lack of significant differences in: (i) the final percentage of germinated seeds of studied species stored at the same conditions, (ii) the final percentage of germinated seeds between the applied stratification conditions in the hybrid and its parental species, and (iii) the mean values of Timson’s index, mean germination time, and coefficient of velocity of germination between the stratification conditions in each species. The statistically significant inter-specific differences in the mean germination time parameter after the +25 °C treatment suggest that the seeds of S. × niederederi are able to germinate faster than the seeds of its parental species. However, to improve our knowledge of naturalization and invasion abilities of S. × niederederi by sexual reproduction, the seed germination and seedling survival of the hybrid should be tested in the field.     

<Emphasis Type="Italic">Luteoamylascus aculeatus</Emphasis> (Pezizomycetes,Pezizaceae): a new genus and species near the <Emphasis Type="Italic">Pachyphlodes</Emphasis>–<Emphasis Type="Italic">Amylascus</Emphasis> lineage

The new taxon Luteoamylascus aculeatus described in this article is proposed to accommodate two collections of a hypogeous ascomycete from central Spain, characterized by a tomentose yellowish peridium, labyrinth-like gleba filled with whitish hyphae, and intensely reacting amyloid asci. ITS, 28S, and RPB2 data suggest that this new taxon is an independent lineage proposed here as the new genus Luteoamylascus. Until now, this lineage was only known from ectomycorrhizal root tips and mitotic spore mats. In phylogenetic analyses, the Luteoamylascus lineage is placed close to the genera Amylascus, Pachyphlodes, and Scabropezia. Morphological data suggest an affinity with Amylascus.     

Genomic constitution of <Emphasis Type="Italic">Festuca</Emphasis> × <Emphasis Type="Italic">Lolium</Emphasis> hybrids revealed by the DArTFest array

Complementary attributes of Festuca and Lolium grasses can be combined in hybrid cultivars called Festuloliums, which are becoming increasingly popular fodder crops and amenity plants. Genomic constitution of commercially available Festuloliums was reported to vary from almost equal representation of parental genomes to apparent lack of one of them based on molecular cytogenetic analyses and screening with a small set of DNA markers, both approaches with limited resolution. Here, we describe the use of the DArTFest array comprising 3,884 polymorphic DArT markers for characterization of genomes in five Festulolium cultivars. In any of the cultivars, the minimum number of informative markers, which discriminated the parental Lolium and Festuca genomes was 361 and 171, respectively. Using the DArTFest array, it was possible to determine hybrid genome constitution at resolution which has never been achieved before and the analysis of a set of randomly selected plants from each cultivar provided information on genetic structure of outcrossing Festulolium cultivars. In addition to a core set of markers typical for each hybrid cultivar, markers occurring at low frequency among the plants within each cultivar were identified. Biological significance of genomic loci associated with the rare markers is yet to be determined. Finally, with the aim to simplify the use of DArTFest arrays to characterize Festuca × Lolium hybrids, various bulking strategies were compared. While all bulks were suitable for identification of hybrids, only bulks of few plants have been found to reveal the rare markers.     

Insoluble but enzymatically active <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

The gene encoding thermostable α-amylase from Bacillus licheniformis consisting of 483 amino acid residues (mature protein) was cloned and expressed in Escherichia coli under the control of T7 promoter. The analysis of the soluble and insoluble fractions after lyzing the host cells revealed that recombinant α-amylase was produced in insoluble aggregates. Despite being produced in the insoluble aggregates the recombinant enzyme was highly active with a specific activity of 408 U/mg.     

Hybridization increases canopy arthropod diversity in the <Emphasis Type="Italic">Quercus affinis</Emphasis> × <Emphasis Type="Italic">Quercus laurina</Emphasis> complex

Understanding the factors that influence the diversity and composition of arthropod communities is a major topic in ecology. Canopy arthropod communities are a major constituent of biodiversity and show great variation in time and space according to different factors. Recently, genetic variation within tree species has attracted attention as a significant factor determining the diversity and composition of canopy arthropod communities. A major source of genetic and phenotypic novelty in plant species is interspecific hybridization, and therefore it is of interest to evaluate how this process affects the communities of associated organisms. In this study, we used microsatellite markers and geometric morphometry of leaf shape to analyze genetic and morphological variation in 45 individuals in a local hybrid zone between the oaks Quercus affinis and Q. laurina in Mexico. Individual trees were assigned to one of the parental species or to the hybrid category. The percentage of leaf area removed by herbivores was quantified in each individual and the canopies of five individuals of each categeory (two parental species and hybrids) was fogged with insecticide to assess the diversity and composition of arthropod communities. Results indicated that hybrid trees experience higher levels of herbivory than parental species and also sustain a higher abundance and richness of canopy arthropods. In general, our study supports the “hybrid susceptibility hypothesis” that predicts a higher incidence of associated arthropods on hybrid plants than in their parental species as result of the disruption of co-adapted gene complexes associated to resistance traits.     

Overexpression of <Emphasis Type="Italic">ARO10</Emphasis> in <Emphasis Type="Italic">pdc5</Emphasis>Δmutant resulted in higher isobutanol titers in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To investigate effects of different pyruvate decarboxylases on isobutanol titers in Saccharomyces cerevisiae, single-gene deletion of the three PDCs genes encoding pyruvate decarboxylases were constructed in this study. In addition, we over-expressed Ilv2, which catalyzed the first step in the valine synthetic pathway, and Bat2, which was the cytoplasmic branched-chain amino-acid aminotransferase that catalyzed L-valine to 2-ketoisovalerate, to increase isobutanol production in the genetically modified strains. Our results showed that knockout of PDC5 were one of the main factors among the three PDC genes for improving isobutanol titers in S. cerevisiae. Additionally, we found that deletion of PDC5 in strain carrying overexpressed ILV2 and ARO10 resulted in 8-fold higher isobutanol productivity as compared to the control strain in micro-aerobic fermentations. Our results also suggested that engineered strain pdc5ΔpILV2 pARO10 generated lower ethanol titers and higher acetate acid titers than the control strain, while the growth rate and glucose consumption rate of engineered strain pdc5ΔpILV2 pARO10 were slightly lower than that of the control strain. Meanwhile, the biomass concentration of pdc5ΔpILV2 pARO10 decreased dramatically than that of the control strain.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.     

<Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>: Significant contemporary hospital pathogen — <Emphasis Type="Italic">review</Emphasis>

Stenotrophomonas maltophilia (Sm) plays an important role as an opportunistic pathogen in immunocompromised individuals. The growing detection rates of this bacterium in hospitalized patients are associated with the invasiveness of therapeutic and diagnostic procedures and the selection pressure of antibiotic therapy. A broad range of infections that can be caused by Sm is frequently bound to biofilm. The high level of intrinsic resistance to many unrelated antibiotics and increasing acquired resistance to the drug of choice, trimethoprim-sulfamethoxazole pose a threat for the near future when our treatment options may become depleted. Prevention of colonization and infection consists in consequent implementation of the rules governing nosocomial infection control, rational use of antibiotics including the optimization of selection and testing of antimicrobial agents suitable for the treatment of stenotrophomonad infections.     

<Emphasis Type="Italic">δ</Emphasis><Superscript>13</Superscript>C values and crassulacean acid metabolism in <Emphasis Type="Italic">Clusia</Emphasis> species from Panama

The genus Clusia is notable in that it contains arborescent crassulacean acid metabolism (CAM) plants. As part of a study of CAM in Clusia, titratable acidities were measured in 25 species and ¹³C values were measured for 38 species from Panamá, including seven undescribed species, and 11 species from Colombia, Costa Rica and Honduras. CAM was detected in 12 species. Clusia flava, C. rosea and C. uvitana exhibited ¹³C values or diurnal fluctuations in acidity indicative of strong CAM. In C. croatii, C. cylindrica, C. fructiangusta, C. lineata, C. odorata, C. pratensis, C. quadrangula, C. valerioi and C. sp. D diurnal fluctuations in acidity were consistent with weak CAM but the ¹³C values were C₃-like. All of the species that exhibited strong or weak CAM were in the C. flava or C. minor species groups. CAM was not detected in any member of the C. multiflora species group. Strong CAM species were not collected at altitudes above 680 m a.s.l. On the basis of ¹³C values, the expression of CAM was similar in terrestrial, hemi-epiphytic and epiphytic species and did not differ between individuals of the same species that exhibited different life-forms. This study indicates that phylogenetic affiliation may be a predictor of an ability to exhibit CAM in Clusia species from the Panamanian region, and that weak CAM is probably a common photosynthetic option in many Clusia species. ¹³C value is not a particularly good indicator of a potential of Clusia species growing in the field to exhibit CAM because it appears that the contribution in most species of CAM to carbon gain is generally rather small when integrated over the life-time of leaves.     

<Emphasis Type="Italic">Adelobotrys atlantica</Emphasis> (<Emphasis Type="Italic">Melastomataceae: Merianieae</Emphasis>): the first species of <Emphasis Type="Italic">Adelobotrys</Emphasis> from Brazil’s Mata Atlântica

Summary  A new species of Adelobotrys DC., A. atlantica Schulman, is described and illustrated. This is the first record of the genus in the Atlantic rainforests of Brazil. The only known specimen of the new species was collected in 1943 close to the sizeable city of Ilhéus in an area that has probably now been converted to managed land. It is therefore possible that A. atlantica is already extinct.