Peroxidase activity against guaiacol,NADH, chlorogenic acid,ferulic acid and coniferyl alcohol in root tips of <Emphasis Type="Italic">Lotus japonicus</Emphasis> and <Emphasis Type="Italic">L. corniculatus</Emphasis> grown under low pH and aluminium stress

The purpose of this study was to examine the impact of low pH and Al stress on the apoplastic production of H₂O₂ and POD activity against guaiacol, ferulic acid, coniferyl alcohol, NADH and chlorogenic acid in the root tip (RT) of two cultivars of Lotus corniculatus and the model Lotus japonicus Gifu, with the goal to determine the possible role of POD activity in proton and Al tolerance. Our results suggest that Lotus corniculatus cv. UFRGS is more tolerant to low pH and Al than cv. Draco due to the high POD activities involved in CW strengthening. The enzymatic response of Lotus japonicus Gifu is similar rather to the sensitive cultivar. On the other hand, in cvs Draco and Gifu low pH induced the activation of CW-modifying PODs, which is probably a component of the defence response of roots to the presence of toxic protons. Aluminium did not activate further these activities suggesting that defence response and acclimation to low pH confers also effective defence against Al toxicity in Lotus species. The activity of NADH-POD and CGA-POD were not affected significantly by exposure to low pH or Al. Al and pH induced drop in H₂O₂ production, which was more relevant in cv. Gifu and Draco than in cv. UFRGS is probably associated with enhanced activity of peroxidases involved in secondary CW metabolism utilizing H₂O₂ as an electron acceptor.     

Two <Emphasis Type="Italic">CLE</Emphasis> genes are induced by phosphate in roots of <Emphasis Type="Italic">Lotus japonicus</Emphasis>

Genes of CLE (CLAVATA3/ESR-related) family encode peptide ligands that regulate plant development in response to external stimuli such as rhizobial infection and the nitrate application as well as various internal stimuli. To investigate whether LjCLE gene(s) may involve in plant response to inorganic phosphate (Pi), we analyzed Pi responses of 39 LjCLE genes in hydroponically grown Lotus japonicus plants (ecotype Miyakojima ‘MG-20’). Two LjCLE genes, LjCLE19 and 20, were up-regulated specifically and greatly in roots of L. japonicus by Pi addition to the hydroponic solution. When the external Pi level increased, expressions of LjCLE19 and 20 increased before the increase in the Pi content in plants. On the other hand, when the external Pi level decreased, the Pi content in plants decreased first, then expression levels of LjCLE19 and 20 decreased. Based on our results, we discuss the relationship between LjCLE19 and 20 and the tissue Pi levels in plants. This is the first report showing induction of specific CLE genes by phosphate.     

Cell-free culture medium of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> improves seed germination and seedling growth in maize (<Emphasis Type="Italic">Zea mays</Emphasis>) and rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

The saprophytic bacterium Burkholderia cepacia has been shown to play an active role as plant growth promoting bacteria (PGPB). In this study, the ability of cell-free culture medium (CFCM) of B. cepacia to improve early developmental stages of plants has been assessed on two agronomically important crops, maize (Zea mays) and rice (Oryza sativa). Treating maize and rice seeds for 45 min before germination significantly improved seed germination and consequent seedling growth. The effect of CFCM was confirmed by the increased biomass of the shoot and, mainly, the root systems of treated seedlings. Chromatographic characterization of the CFCM revealed that the spent culture medium of B. cepacia is a complex mix of different classes of metabolites including, among others, salicylic acid, indole-3-acetic acid (IAA) and several unidentified phenolic compounds. Fractionation of the CFCM components revealed that the impressive development of the root system of CFCM-treated seedlings is due to the synergistic action of several groups of components rather than IAA alone. The data presented here suggest that a CFCM of B. cepacia can be used to improve crop germination.     

Effect of Plant Growth-Promoting Rhizobacteria and Culture Filtrate of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> on Phenolic and Salicylic Acid Contents in Chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis>)

Two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens strain Pf4 and P. aeruginosa strain Pag, protected chickpea (Cicer arietinum) plants from Sclerotium rolfsii infection when applied singly or in combination as seed treatment. Pag gave the best protection to the seedlings, applied either singly (mortality 16%) or in combination with Pf4 (mortality 17%) compared with 44% and 24% mortality in control and Pf4 treatment, respectively. The two PGPR strains induced the synthesis of specific phenolic acids, salicylic acid (SA), as well as total phenolics at different growth stages of chickpea seedlings with varied amount. The maximum amount of total phenolics was recorded in all the aerial parts of 4-week-old plants. Gallic, ferulic, chlorogenic, and cinnamic acids were the major phenolic acids detected in high-performance liquid chromatography (HPLC) analysis. Induction of such phenolic acids in the seedlings was observed up to 6 weeks in comparison with control. Salicylic acid (SA) was induced frequently during the first 3 weeks of growth only. Between the two strains, Pag was more effective in inducing phenolic acid synthesis applied either singly or in combination with strain Pf4 during the entire 6 weeks of growth of chickpea. In the presence of a culture filtrate of S. rolfsii, the two Pseudomonas strains induced more phenolic acids in treated than in non-treated and control plants. The occurrence of salicylic acid was frequent in the first 24 h, but infrequent at 48 and 96 h. Foliar spray of Pseudomonas strains also enhanced the phenolic acid content as well as total phenolics within 24 h of application. Gallic, chlorogenic, and cinnamic acids were consistently discerned in the treated leaves, whereas SA was absent even up to 96 h of application. Resistance in chickpea plants by Pseudomonas strains through induction of phenolic compounds as well as induced systemic resistance via SA-dependent pathway was evident. Received: 1 April 2002 / Accepted: 4 May 2002     

Decolourisation and dephenolisation potential of selected <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Nigri</Emphasis> strains – <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> in olive mill wastewater

Aspergillus section Nigri strains Aspergillus aculeatus Ege-K 258, A. foeditus var. pallidus Ege-K156, A. niger Ege-K 4 and A. tubingensis Ege-K 265 were used to treat olive mill wastewater (OMW) in an investigation aimed at exploring their dephenolisation and decolourisation ability and, consequently, the economic feasibility of using any or all of these strains in a pre-treatment step in the processing of OMW. Of these strains A. tubingensis Ege-K 265 resulted in an 80% decolourisation of twofold-diluted OMW and a 30% decolourisation of undiluted OMW; in addition, it was able to remove approximately 30% of all phenolic compounds in both twofold-diluted and undiluted OMW. We conclude that A. tubingensis Ege-K 265 could be effectively used in the pre-treatment step of a combined aerobic-anaerobic process to solve the environmental problems caused by OMW in Mediterranean countries.     

The <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> secondary metabolite 2,4 diacetylphloroglucinol impairs mitochondrial function in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Pseudomonas fluorescens strains are known to produce a wide range of secondary metabolites including phenazines, siderophores, pyoluteorin, and 2,4 diacetylphloroglucinol (DAPG). DAPG is of particular interest because of its antifungal properties and because its production is associated with inhibition of phytopathogenic fungi in natural disease-suppressive soils. This trait has been exploited to develop strains of P. fluorescens that have potential application as biocontrol agents. Although the biochemistry, genetics and regulation of DAPG production have been well-studied, relatively little is known about how DAPG inhibits fungal growth and how fungi respond to DAPG. Employing a yeast model and a combination of phenotypic assays, molecular genetics and molecular physiological probes, we established that inhibition of fungal growth is caused by impairment of mitochondrial function. The effect of DAPG on yeast is largely fungistatic but DAPG also induces the formation of petite cells. Expression of the multidrug export proteins Pdr5p and Snq2p is increased by DAPG-treatment but this appears to be a secondary effect of mitochondrial damage as no role in enhancing DAPG-tolerance was identified for either Pdr5p or Snq2p.     

Structural characteristics of the chloroplast <Emphasis Type="Italic">rpS16</Emphasis> intron in <Emphasis Type="Italic">Allium sativum</Emphasis> and related <Emphasis Type="Italic">Allium</Emphasis> species

The intron sequence of chloroplast rpS16 and the secondary structure of its pre-mRNA were characterized for the first time in 26 Allium sativum accessions of different ecologo-geographical origins and seven related Allium species. The boundaries and main stem-loop consensus sequences were identified for all six domains of the intron. Polymorphism was estimated for the total intron and its regions. The structural regions of the rpS16 intron proved to be heterogeneous for mutation rate and spectrum. Mutations were most abundant in domains II and IV, and transition predominated in domains I, III, V, and VI. In addition to structural elements and motifs typical for group IIB introns, several Allium-specific micro- and macrostructural mutations were revealed. A 290-bp deletion involving domains III and IV and part of domain V was observed in A. altaicum, A. fistulosum, and A. schoenoprasum. Several indels and nucleotide substitutions were found to cause a deviation of the pre-mRNA secondary structure from the consensus model of group II introns.     

Revision of “<Emphasis Type="Italic">Septonema ochraceum</Emphasis>” revealed three new species of <Emphasis Type="Italic">Venturiaceae</Emphasis> and <Emphasis Type="Italic">Herpotrichiellaceae</Emphasis>

Survey of seven strains determined as Septonema ochraceum (Dothideomycetes, inc. sed.) isolated from pine litter or obtained from public collections revealed three new species, Fusicladium cordae, F. sicilianum (Venturiaceae), Cladophialophora matsushimae (Herpotrichiellaceae) and a cryptic species morphologically identical to Devriesia americana (Teratosphaeriaceae), but phylogenetically distinct. Morphological survey and phylogenetic analysis using nucleotide sequence data from the nuclear ribosomal subunit genes indicate a close relationship within three species colonising pine litter needles, F. cordae, F. pini and F. ramoconidii. F. sicilianum is most related to F. rhodense. C. matsushimae represents a species belonging to one of the lineages of the polyphyletic genus Cladophialophora. None of the strains observed can be classified morphologically as S. ochraceum, of which the type material does not exist.     

Comparative analysis of <Emphasis Type="Italic">iol</Emphasis> clusters in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> strains

The present study was designed to expand genetic knowledge of myo -inositol (MI) metabolism in Lactobacillus casei. Twenty-four L. casei isolates of dairy origin were tested for the presence of iol cluster. PCR screening revealed eight strains encoded functions involved in MI utilization, of which one strain was able to use MI as carbon source. To gain a deeper understanding of the function of iol genes, four of the eight observed iol clusters were subjected to the full sequencing procedure. The results showed that the iol cluster was not a common feature among dairy L. casei strains. In addition, the four iol clusters were highly similar to one another in terms of sequence similarity and operon architecture. However, abundant polymorphisms that comprised a majority of synonymous mutations were detected throughout the full sequences. Three of them distributed among iolB, iolC, and iolT genes were found in linkage to MI-negative phenotype. Compared with other bacterial iol clusters, the iol cluster of L. casei showed a high similarity with that of Bacillus subtilis.     

Influence of <Emphasis Type="Italic">Daphnia</Emphasis> infochemicals on functional traits of <Emphasis Type="Italic">Microcystis</Emphasis> strains (Cyanobacteria)

We conducted a laboratory experiment to investigate the influence of Daphnia infochemicals on growth rate, microcystin production, colony formation and cell size of eight Microcystis strains isolated from two lakes. The strains were characterized genetically by their 16S-23S rDNA ITS sequence. The experiment was composed of four treatments: (1) a control using filtered WC medium, (2) addition of Scenedesmus obliquus culture medium filtrate, (3) addition of Daphnia magna culture medium filtrate and (4) addition of sodium octyl sulphate, a commercially available Daphnia infochemical. Our results showed that sympatric strains differed strongly for the measured functional traits, while no correlations between traits were found. Between-strain differences in growth rate, microcystin production, colony formation and cell size were generally larger than the differences in phenotypes observed between treatments. Despite this, several strains reacted to the infochemicals by changing functional trait values. Daphnia culture medium filtrate and, to a lesser extent, sodium octyl sulphate had a negative influence on the growth rate of half of the strains and stimulated microcystin production in one strain, but the latter effect was not Daphnia-specific as Scenedesmus culture medium filtrate had the same effect. Daphnia culture medium filtrate also induced colony formation in one strain. Our data suggest that Daphnia infochemicals generally have a weak influence on growth rate, microcystin production and colony formation of Microcystis strains as compared to the inter-strain variability, while existing inducible effects are highly strain-specific.     

Interaction of <Emphasis Type="Italic">Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Impact of low pH and aluminium on nitrogen uptake and metabolism in roots of <Emphasis Type="Italic">Lotus japonicus</Emphasis>

The effect of low pH and aluminum on nitrogen uptake and metabolism was studied in roots of Lotus japonicus grown in hydroponic cultures. The low pH slightly suppressed root elongation, and this effect was accompanied by the suppression of nitrate and ammonia uptake, as well as the nitrate reductase activity. In spite of high resistance of young Lotus plants to short-term Al application, the one-day treatment of Al strongly reduced nitrate uptake and also the activity of nitrate reductase (NRA) in the apical parts of roots. The glutamine synthetase activity was also suppressed by Al treatment, but in lower extent. On the other hand, the ammonium uptake and nitrite reductase activity stayed unchanged by Al treatment and the values were practically the same as in control plants. These results support the view that nitrate uptake and nitrate reduction might be the main processes responsible for Al induced growth retardation in Lotus plants grown in mineral acid soils.     

High-Level Expression of Heme-Dependent Catalase Gene <Emphasis Type="Italic">katA</Emphasis> from <Emphasis Type="Italic">Lactobacillus Sakei</Emphasis> Protects <Emphasis Type="Italic">Lactobacillus Rhamnosus</Emphasis> from Oxidative Stress

Lactic acid bacteria (LAB) are generally sensitive to hydrogen peroxide (H₂O₂), Lactobacillus sakei YSI8 is one of the very few LAB strains able to degrade H₂O₂ through the action of a heme-dependent catalase. Lactobacillus rhamnosus strains are very important probiotic starter cultures in meat product fermentation, but they are deficient in catalase. In this study, the effect of heterologous expression of L. sakei catalase gene katA in L. rhamnosus on its oxidative stress resistance was tested. The recombinant L. rhamnosus AS 1.2466 was able to decompose H₂O₂ and the catalase activity reached 2.85 μmol H₂O₂/min/10⁸ c.f.u. Furthermore, the expression of the katA gene in L. rhamnosus conferred enhanced oxidative resistance on the host. The survival ratios after short-term H₂O₂ challenge were increased 600 and 10⁴-fold at exponential and stationary phase, respectively. Further, viable cells were 100-fold higher in long-term aerated cultures. Simulation experiment demonstrated that both growth and catalase activity of recombinant L. rhamnosus displayed high stability under environmental conditions similar to those encountered during sausage fermentation.     

<Emphasis Type="Italic">Pichia anomala</Emphasis> in grain biopreservation

Cereal grain is a major component of food and feed in large parts of the world. The microbial flora on cereal grains may interfere with hygiene and storage stability, palatability and bioavailability of minerals and proteins may depend on the composition of the microbial population. Therefore, it is of primary interest to control the microbial species present on cereal grain. Inoculation of the biocontrol yeast Pichia anomala to cereal feed grain improved feed hygiene by reduction of moulds and Enterobacteriaceae, and enhanced the nutritional value by increasing the protein content and reducing the concentration of the antinutritional compound phytate. P. anomala strains showed a high phytase activity, for some strains also considerable extracellular phytase activity was observed. A certain maximum in biomass concentration was never exceeded indicating cell density induced growth inhibition of P. anomala.     

Impact of heat-inactivated <Emphasis Type="Italic">Lactobacillus casei</Emphasis> and <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> strains on cytokine responses in whole blood cell cultures of children with atopic dermatitis

Heat-inactivated Lactobacillus casei LOCK 0900, L. casei LOCK 0908 and Lactobacillus paracasei LOCK 0919 strains, applied to blood cell cultures obtained from children with atopic dermatitis induced production of anti-allergic T_H1 cytokines (interleukin-12, interleukin-18, interferon-γ, tumor necrosis factor-α) and regulatory transforming growth factor-β₁), but did not stimulate pro-allergic interleukin-5. The lactobacilli-mixture remarkably enhanced the T_H1 response compared to single strains. This synergistic effect was not observed for transforming growth factor-β₁. In contrast, the amount of interleukin-10 was found to be considerably lower when cells were stimulated with lactobacilli-mixture compared to single strains. The mixture of Lactobacillus strains represents a probiotic bacterial preparation modulating in vitro cytokine profile of allergic children towards anti-allergic T_H1 response.     

NO- and H<Subscript>2</Subscript>S-synthesizing neurons in the thoracic ganglion of crabs <Emphasis Type="Italic">Eriocheir japonicus</Emphasis> and <Emphasis Type="Italic">Pugettia quadridens</Emphasis> (Decapoda)

The presence and distribution of NO and H₂S-synthesizing elements in the thoracic ganglion in two crab species, Eriocheir japonicus and Pugettia quadridens, are shown using the staining methods of NADPH-diaphorase histochemistry and cystathionine-β-synthase (CBS) immunohistochemistry. NO and H₂S have been established to be synthesized by different neurons in the thoracic ganglion. The high CBS activity in efferent elements (segmental motoneurons, interneurons and nerve fibers in the limb neuropil) indicates the involvement of hydrogen sulfide in the organization of locomotor behavior in crustaceans. The presence of H₂S-synthesizing neurons in the wall of the sternal artery in E. japonicus allows suggesting the participation of H₂S in the regulation of vascular tone.     

Early antibody response against <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">paratuberculosis</Emphasis> antigens in subclinical cattle

Background  

Our laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection.     

A role of <Emphasis Type="Italic">ygfZ</Emphasis> in the <Emphasis Type="Italic">Escherichia coli</Emphasis> response to plumbagin challenge

Plumbagin is found in many herbal plants and inhibits the growth of various bacteria. Escherichia coli strains are relatively resistant to this drug. The mechanism of resistance is not clear. Previous findings showed that plumbagin treatment triggered up-regulation of many genes in E. coli including ahpC, mdaB, nfnB, nfo, sodA, yggX and ygfZ. By analyzing minimal inhibition concentration and inhibition zones of plumbagin in various gene-disruption mutants, ygfZ and sodA were found critical for the bacteria to resist plumbagin toxicity. We also found that the roles of YgfZ and SodA in detoxifying plumbagin are independent of each other. This is because of the fact that ectopically expressed SodA reduced the superoxide stress but not restore the resistance of bacteria when encountering plumbagin at the absence of ygfZ. On the other hand, an ectopically expressed YgfZ was unable to complement and failed to rescue the plumbagin resistance when sodA was perturbed. Furthermore, mutagenesis analysis showed that residue Cys228 within YgfZ fingerprint region was critical for the resistance of E. coli to plumbagin. By solvent extraction and HPLC analysis to follow the fate of the chemical, it was found that plumbagin vanished apparently from the culture of YgfZ-expressing E. coli. A less toxic form, methylated plumbagin, which may represent one of the YgfZ-dependent metabolites, was found in the culture supernatant of the wild type E. coli but not in the ΔygfZ mutant. Our results showed that the presence of ygfZ is not only critical for the E coli resistance to plumbagin but also facilitates the plumbagin degradation.