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 <Emphasis Type="Italic">Azotobacter chroococcum</Emphasis> on <Emphasis Type="Italic">in vitro</Emphasis> pineapple plants’ growth during acclimatization

Pineapple is one of the most important tropical fruits, but the availability of planting material is insufficient to agricultural demands. Therefore, several pineapple micropropagation protocols have been developed. However, acclimatization of in vitro plants continues to take a prolonged period. Biofertilizers have been found as safe alternatives to improve the agricultural performances of many crops. This study highlights some of the effects of the application of Azotobacter chroococcum (INIFAT5 strain) on in vitro pineapple plants during acclimatization. The bacteria were sprayed immediately after transplanting to the ex vitro environment; the plants were then sprayed every 4 wk. A control group of plants was established. Subsequently, after 5 mo, the evaluated variables included fresh and dry plant weight, plant height (cm), and root length (cm). The anatomy of middle-aged leaves and roots was also studied: transversal thickness and width of cuticle, epidermis, hypodermis, aquiferous parenchyma, and photosynthetic parenchyma. Thickness of root exoderm, external cortex, internal cortex, and stele were also evaluated. In general, the INIFAT5 strain improved the plant development. Results showed that the bacteria significantly provoked changes in the plant fresh weight, the thickness of the leaf abaxial and adaxial cuticles, and the root exoderm width. Contrastingly, A. chroococcum did not affect the thickness of the leaf photosynthetic parenchyma.     

Mapping species differences for adventitious rooting in a <Emphasis Type="Italic">Corymbia torelliana</Emphasis> × <Emphasis Type="Italic">Corymbia citriodora</Emphasis> subspecies <Emphasis Type="Italic">variegata</Emphasis> hybrid

Quantitative trait loci (QTL) detection was carried out for adventitious rooting and associated propagation traits in a second-generation outbred Corymbia torelliana × Corymbia citriodora subspecies variegata hybrid family (n = 186). The parental species of this cross are divergent in their capacity to develop roots adventitiously on stem cuttings and their propensity to form lignotubers. For the ten traits studied, there was one or two QTL detected, with some QTL explaining large amounts of phenotypic variation (e.g. 66% for one QTL for percentage rooting), suggesting that major effects influence rooting in this cross. Collocation of QTL for many strongly genetically correlated rooting traits to a single region on linkage group 12 suggested pleiotropy. A three locus model was most parsimonious for linkage group 12, however, as differences in QTL position and lower genetic correlations suggested separate loci for each of the traits of shoot production and root initiation. Species differences were thought to be the major source of phenotypic variation for some rooting rate and root quality traits because of the major QTL effects and up to 59-fold larger homospecific deviations (attributed to species differences) relative to heterospecific deviations (attributed to standing variation within species) evident at some QTL for these traits. A large homospecific/heterospecific ratio at major QTL suggested that the gene action evident in one cross may be indicative of gene action more broadly in hybrids between these species for some traits.     

Selecting Bacterial Strains for Use in the Biocontrol of Diseases Caused by <Emphasis Type="Italic">Phytophthora capsici</Emphasis> and <Emphasis Type="Italic">Alternaria alternata</Emphasis> in Sweet Pepper Plants

More than 500 isolates of bacteria were obtained from the aerial part and rhizosphere of sweet pepper (Capsicum annuum L.) plants harvested from different places in the Region of Murcia (Spain). The isolates were purified and assayed in vitro against Phytophthora capsici and Alternaria alternata. Sixty isolates (12 %) produced an inhibition zone against at least one of the pathogens, while ten had a strongly inhibitory effect on both pathogens assayed. Microscopic observation of interactions zone showed cell vacuolisation, hyphae lysis and spilling of cytoplasm content of the pathogens in the culture media. These ten isolates were then chosen for biocontrol of Phytophthora root rot and Alternaria leaf spots of pepper plants in vivo. Four of them denominated HS93, LS234, LS523 and LS674 reduced P. capsici root rot by 80, 51, 49 and 54 %, respectively, and A. alternata leaf spots by 54, 74, 62 and 53 %. HS93 belongs to the genus Bacillus and probably the species subtilis, while LS234, LS523 and LS674 belong to the genus Bacillus and probably the species licheniformis. Dry mass of plants treated with these bacteria was significantly higher than that of non-treated and inoculated plants.     

Anatomical observation of polyphenol changes in epidermal cells during the development of <Emphasis Type="Italic">Quercus acutissima</Emphasis>–<Emphasis Type="Italic">Scleroderma verrucosum</Emphasis> ectomycorrhizae

Quercus acutissima seedlings were cultivated in growth pouches and inoculated with Scleroderma verrucosum in order to assess the changes in polyphenol contents in epidermal cells during ECM development. Semithin sections stained with metachromatic Toluidine Blue O (TBO) were compared among non-inoculated lateral roots, early mantled lateral roots, and mycorrhizal roots with a mature mantle. Hyphae adhered closely or were embedded in mucilage-like materials on the epidermis. Epidermal cells and root hairs of the non-inoculated second-order lateral roots developing from the taproot harbored polyphenolic compounds that were stained by TBO. At non-inoculated stage, the average numbers of epidermal cells stained entirely (PC2), stained partially (PC1) or remaining unstained (PC0) were 16.5 ± 0.7, 0 ± 0, and 0 ± 0, respectively. At the early mantled stage, the numbers were 6.5 ± 1.6, 5.2 ± 1.4, and 4.2 ± 1.0, and at the mycorrhizal stage, it was 0 ± 0, 0 ± 0, and 32.8 ± 1.3 for PC2, PC1, and PC0, respectively. Total phenolic content in the root tips at each developmental stage declined with ECM development. The early mantled stage involved a dynamic process of polyphenol localization. However, some epidermal cells and endodermal cells of the proximal zone accumulated polyphenols. Eventually, polyphenolic compounds, which were found abundantly in the epidermal cells and root hairs of the non-inoculated lateral roots of the host, disappeared at the mycorrhization process with the symbiont.     

Immunological and biological relationship among flagellin of <Emphasis Type="Italic">Pseudomonas aeruginosa,Burkholderia cepacia</Emphasis>, and <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>

The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.     

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.     

Chemical composition and antimicrobial activity of <Emphasis Type="Italic">Thymus pannonicus</Emphasis> All. (<Emphasis Type="Italic">Lamiaceae</Emphasis>) essential oil

This paper presents the results of a study on chemical composition and antimicrobial activity of Thymus pannonicus All. (Lamiaceae) essential oil from Vojvodina province (north of Serbia). The investigated oil was hydrodistilled from a flowering plant and analysed by GC and GC-MS. Fifty-three constituents were identified (>97% of total oil), with geranial (41.42%, w/w) and neral (29.61%, w/w) as the most prominent. The antimicrobial activity of the oil was evaluated using agar disc diffusion and broth microdilution method against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, two strains of Klebsiella pneumoniae and two strains of Candida albicans. The essential oil exhibited antimicrobial activity to varying degrees against all tested strains. The maximum activity of T. Pannonicus oil was observed against E. coli, S. aureus and both tested strains of C. Albicans (MIC = 50 μ/ml, each). Moderate activity was observed against P. aeruginosa and one of the tested strains of K. Pneumoniae (MIC = 200 μ/ml), while E. faecalis and the other strain of K. Pneumoniae expressed a higher degree of resistance (MIC > 200 μ/ml). This study confirms that essential oil of T. pannonicus possesses remarkable in vitro antimicrobial activity against several medicinally important pathogens. This is attributable to lemon-scented citral, a mixture of geranial and neral, which has well-documented antimicrobial activity against a range of bacteria and fungi.     

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">In vitro</Emphasis> propagation and secondary metabolites production in wild germander (<Emphasis Type="Italic">Teucrium polium</Emphasis> L.)

A protocol for in vitro propagation of the wild germander (Teucrium polium L.) was developed. In vitro plants were developed from ex vitro axillary buds. Then, shoot tips were excised and established on Murashige and Skoog medium. Proliferation of shoots was tested with different levels of 6-furfurylaminopurin, 6-benzyladenine, or thiadiazuron. The highest proliferation of T. polium was obtained when 6-benzyladenine and 6-furfurylaminopurin were used at 2.0 and 1.6 mg l⁻¹, respectively. Thiadiazuron gave the lowest response for shoot proliferation. Rooting was experimented at different levels of Indol-3-butric acid, Indol-3-acetic acid, or 1-naphthaleneacetic acid. 1-Naphthaleneacetic was the only growth regulator which promoted root induction. Rooted plants were acclimatized successfully with 75% survival and grown in the greenhouse. In vitro- and in vivo-grown plants were analyzed for essential oil production. In vitro-grown T. polium on MS medium supplemented with 6-benzyladenine and 1-naphthaleneacetic gave higher oil yield than that grown on hormone-free Murashige and Skoog medium. In vivo (wild)-grown T. polium produced different oil yield when collected in different months (April and October). β-caryophyllene, used as a marker compound in the essential oil, was identified and quantified by gas chromatography (GC) analysis. Gas chromatography/mass (GC-MS) spectrometry analysis was also used to identify other components of in vitro cultures and to compare with in vivo-grown plants.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the plant pathogenic fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern b lot analy s is. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.     

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.     

Identification of phenotypically and genotypically related <Emphasis Type="Italic">Lactobacillus</Emphasis> strains based on nucleotide sequence analysis of the <Emphasis Type="Italic">groEL,rpoB, rplB</Emphasis>, and <Emphasis Type="Italic">16S rRNA</Emphasis> genes

Sixty-eight cultures of lactic acid bacteria were isolated and identified from national fermented milk drinks (airan, koumiss, kurunga, shubat) home-made in different regions of the Republic of Kazakhstan and the Buryat Republic of Russia. The cultures of lactic acid bacteria of the genus Lactobacillus were identified as L. paracasei and L. rhamnosus related to the L. casei group and as L. brevis, L. buchneri, L. diolivorans, and L. parabuchneri (the L. buchneri group) using the classical microbiological methods and on the basis of the 16S rRNA gene sequence analysis. The polymorphism of the nucleotide sequences of the genes groEL, rpoB, and rplB encoding specific proteins was studied for intraspecific differentiation of the lactobacilli. The analysis of these genes allowed a more accurate identification of the lactobacilli that are genetically and phenotypically related to the L. casei group as L. paracasei subsp. paracasei and L. paracasei subsp. tolerans. The gene nucleotide sequences of all the genotyped strains were deposited in the GenBank database.     

Habitat,density, and spatial distribution of <Emphasis Type="Italic">Rivulus giarettai</Emphasis> (<Emphasis Type="Italic">Actinopterygii</Emphasis>, <Emphasis Type="Italic">Cyprinodontiformes</Emphasis>) in southeastern Brazil

We present data on the habitat, density, and spatial distribution of Rivulus giarettai, and discuss some biotic and abiotic variables related to its abundance in Free Flowing Waters (FFW) and Dam Reservoirs (DR) in palm grove (Mauritia flexuosa) marshes (Veredas) in Uberlandia, Minas Gerais, southeastern Brazil. The mean density (individuals/plot) of R. giarettai was about 13 times higher in FFW than in DR. In FFW, the density of R. giarettai was highest at intermediate amounts of substrate (plant mass) and it was positively rank-correlated with the depth, and the number of arthropods. Individuals occurred in an aggregated distribution. The aggregated pattern could be related to a concentration of individuals in microhabitats neither too much exposed nor completely saturated by plants. R. giarettai was relatively abundant and tolerant to slight man-made habitat modifications. Damming appeared to be especially problematic by negatively affecting its density.     

Effects of sugars and growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> growth of <Emphasis Type="Italic">Dactylorhiza</Emphasis> species

The influence of sugars and growth regulators on shoot and root growth of Dactylorhiza species was studied under in vitro conditions. The seedling development was stimulated with the application of glucose and sucrose at concentration of 10 g dm⁻³ each. The improvement of shoot growth rate and shoot length was enhanced by cytokinins N ⁶-(2-isopentenyl)adenine or N ⁶-benzyladenine and their combination with auxin indolebutyric acid (IBA). The root growth rate and root length of seedlings increased in the presence of IBA and α-naphthaleneacetic acid. Individual Dactylorhiza species showed statistically significant differences in shoot and root development depending on sugar and growth regulator combinations.