Root-promoting rhizobacteria in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> cuttings

Cloned Eucalyptus spp. plantations are based in greenhouse production of plants generated by vegetative propagation. Diverse studies have demonstrated that rhizospheric bacteria can stimulate plant growth, and more recently that they can increase rooting in vegetative material. Considering this potential, the objective of this study was to verify the effect of bacterial strains on rooting Eucalyptus globulus. A total of 132 bacterial strains isolated from the rhizosphere of E. globulus and Eucalyptus nitens were studied. The bacterial inoculums in a concentration of 4 × 10⁸ cfu/ml were applied to the rooting substrate at the cutting installation and 45 days after by irrigation. Rooting was evaluated on days 60 and 75 after cutting installation, considering the number of roots as well as their fibrosity and roots biomass. Of the 132 strains evaluated, 26 significantly increased cutting rooting in a range of 191.4–69.4% with respect to the control. Additionally, some strains stimulated the development of fine roots and incremented the roots biomass. The strains identificated that produced a rooting effect were: Bacillus firmus, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, B. subtilis/amyloliquefaciens, Bacillus circulans, Brevibacillus brevis, Paenibacillus lautus and Stenotrophomona maltophilia. These first trials suggest the potential of these bacteria to be used in clonal production programs for E. globulus.     

Genomic predictions of breeding values in a cloned <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> population in Chile

In Chile, an intensive Eucalyptus globulus clonal selection program is being carried out to increase forest productivity for pulp production. A breeding population was used to investigate the predicted ability of single nucleotide polymorphism (SNP) markers for genomic selection (GS). A total of 310 clones from 53 families were used. Stem volume and wood density were measured on all clones. Trees were genotyped at 12 K polymorphic markers using the EUChip60K genotype array. Genomic best linear unbiased prediction, Bayesian lasso regression, Bayes B, and Bayes C models were used to predict genomic estimated breeding values (GEBV). For cross-validation, 260 individuals were sampled for model training and 50 individuals for model validation, using 2 folds and 10 replications each. The average predictive ability estimates for wood density and stem volume across the models were 0.58 and 0.75, respectively. The average rank correlations were 0.59 and 0.71, respectively. Models produced very similar bias for both traits. When clones were ranked based on their GEBV, models had similar phenotypic mean for the top 10% of the clones. The predicted ability of markers will likely decrease if the models are used to predict GEBV of new material coming from the breeding program, because of a different marker–trait phase introduced by recombination. The results should be validated with larger populations and across two generations before routine applications of GS in E. globulus. We suggest that GS is a viable strategy to accelerate clonal selection program of E. globulus in Chile.     

Genotoxic activity of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> essential oil in <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> diploid cells

Eucalyptus globulus essential oil was evaluated for its genotoxic potential using a somatic segregation assay and a diploid strain of the fungus Aspergillus nidulans, heterozygous for nutritional and conidia color markers. The main compounds of the current essential oil sample were eucalyptol (49.0 %), α-pinene (8.9), β-pinene (1.5), globulol (6.9), α-eudesmol (1.12), spathulenol (1.42), γ-cadinene (1.45), trans-β-elemenone (1.23) and aromandendrene (2.3), totaling 74 % of oil. Oil at 0.12 and 0.25 μL/mL was found to increase the mitotic instability of the original diploid strain and the number of diploid mitotic recombinants of A. nidulans. The genotoxicity of the oil was associated with the induction of mitotic crossing-over or with oil-broken chromosomes.     

Regeneration patterns of <Emphasis Type="Italic">Polylepis subtusalbida</Emphasis> growing with the exotic trees <Emphasis Type="Italic">Pinus radiata</Emphasis> and <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> at Parque Nacional Tunari,Bolivia

Dry semideciduous and deciduous forests occur only on calcium-rich soils and occupy almost 20% of the 200 million ha of the cerrado region of central Brazil. Other savanna physiognomies of woodlands and grasslands, and gallery forests generally occur on acid soils with low calcium levels. The literature on phytosociological aspects of such cerrado physiognomies is quite abundant whereas there is very little information on deciduous forests. Also lacking is information on soil fertility and its influence on species distribution. The objective of the present study was to study the distribution of native species within a dry deciduous forest in the Araguari river valley, Uberlândia, Minas Gerais as related to soil properties. A 1-ha sampling area, with no apparent difference in physiognomy, was divided into fifty 10 m × 20 m plots arrayed in 10 × 5 grid and all trees with a minimum circumference of 15 cm at 1.30 m height were surveyed. A composite soil sample was collected from 0 to 10 cm depth in each plot and soil pH, organic C and nutrient availability were determined. Cluster analysis based on soil chemical properties showed the existence of two distinct groups of plots. Further field examinations revealed that the forest was situated in an area of transition between mica schist and basalt parent materials, without any apparent difference in the forest physiognomy. The basalt-derived soil in 23 plots had significantly higher availability of Ca (47.0 cmol(+) kg^?1 soil), Mg (2.59), and K (0.96) than the mica schist derived soil (Ca—18.4, Mg—1.29, and K—0.66) in the remaining 27 plots, though both soils would be classified as eutrophic. A total of 59 species belonging to 27 families were encountered in the area as a whole, of which 36 were common to both soils. In total, 16 species occurred exclusively on the mica schist soil and 7 on the basaltic soil. Myracrodruon urundeuva Allem. (Anacardiaceae) and Tabebuia roseo-alba (Ridl.) Sandwith (Bignoniaceae) were species with high importance values in the forest as a whole. Though there was no significant difference in the diversity index between the two soils, the importance values of these species was larger in the basalt-derived soil with higher nutrient availability.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Recovery after defoliation in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> saplings: respiration and growth

Key message

Recovery after partial defoliation and/or debudding treatments was found to be more closely related to the release of latent buds rather than temporal changes in leaf-level respiration and carbon uptake.

Abstract

Despite the importance of respiration in the overall carbon balance of plants, recovery after defoliation and debudding has been largely related to changes in carbon uptake; the significance of respiration has received much less attention. Growth, biomass and leaf-level carbon balance (both photosynthesis and dark respiration at night) responses of young Eucalyptus globulus potted-saplings to debudding (B), partial defoliation (D) and combined B&D treatments were assessed over a 12-week recovery period. Light-saturated photosynthetic rates (A ₁₅₀₀) were asynchronous with night respiration rates (R _dark) throughout the course of the experiment; 5 weeks after defoliation, significant increases in A ₁₅₀₀ were accompanied by concomitant increases in R _dark in the B&D and B and D treatments. By week 8, while A ₁₅₀₀ returned to control values, R _dark had increased, particularly in the B&D treatment. Saplings in the B and D treatments showed full recovery with growth, biomass and leaf area being similar to control saplings by week 12. In contrast, saplings in the B&D treatment appeared unable to compensate for the combined removal of all buds and 35 % leaf area as evidenced by slowed height increments and reductions in total biomass of >30 %. Simple modelling of whole-plant net CO₂ uptake showed that saplings in the B&D treatment fixed 20 % less CO₂ than the other treatments at week 12, suggesting that recovery following this treatment and the D treatment was dependent on changes in total leaf area development and whole-tree assimilation rather than differences in assimilation or respiration per unit foliage area. Increased biomass allocation to bud in weeks 5 and 8 suggested that the pattern of refoliation after defoliation and debudding was related to changes in tree architecture from the release of latent buds.

     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Benefits of <Emphasis Type="Italic">Helicobacter pylori</Emphasis><Emphasis Type="Italic">cagE</Emphasis> genotyping in addition to <Emphasis Type="Italic">cagA</Emphasis> genotyping: a Bulgarian study

Associations of Helicobacter pylori cagE status with complex patient characteristics remain to be elucidated in Eastern Europe. The aim of this study was to assess the frequencies of cagE gene and cagA/cagE combinations in H. pylori strains from symptomatic Bulgarian patients and to improve cagA detection. cagA and cagE genotypes were evaluated in 219 patients with single-strain infections. In total, 84.9% of strains were cagA ⁺, while 68.5% were cagE ⁺. cagA ⁺, cagE ⁺, and cagA ⁺/cagE ⁺ strains were more prevalent in peptic ulcer (93.8%, 84.4%, and 84.4%) compared with nonulcer patients (81.3%, 61.9%, and 61.3%, respectively). In elderly patients, cagE ⁺ and cagA ⁺/cagE ⁺ strains were 1.9-fold more common than in the 12 children evaluated. Only 10% of the elderly subjects harbored low-virulence cagA ⁺/cagE ⁻ strains compared with 16.8% of adults and 41.7% of children. Intriguingly, prevalence of the cagA ⁺/cagE ⁻ genotype was 2.1-fold lower in men than in women, suggesting a higher frequency of more virulent strains in men. The presence of both cag genes and combinations was not linked to strain susceptibility to clarithromycin or metronidazole, place of residence, or prior therapy. Use of an extra primer pair increased cagA detection in 14.7% of 31 cagA ⁻ strains. In conclusion, use of a second primer pair for the cagA gene can be recommended in countries with common cagA ⁺ strains. Although both cag genes were linked to severe diseases in Bulgarian patients, the best discrimination of virulent strains was obtained by the cagA/cagE combination or by the cagE gene alone. cagE prevalence increased gradually with patient age, while the cagA ⁺/cagE ⁻ genotype, implying a disrupted cag pathogenicity island, was associated with both younger age and female gender.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Combined effects of climate,habitat, and disturbance on seedling establishment of <Emphasis Type="Italic">Pinus pinaster</Emphasis> and <Emphasis Type="Italic">Eucalyptus globulus</Emphasis>

The natural expansion of forestry trees into habitats outside plantations is a concern for managers and conservationists. We studied seedling emergence and survival of the two main forestry species in Portugal: Eucalyptus globulus (exotic) and Pinus pinaster (native); using a seed addition experiment. Our main objective was to evaluate the combined effects of climate (mild-summer and warm-summer climate), habitat (oak forest and shrubland), and disturbance (vegetation removal and non-disturbance) on the seedling establishment of species in semi- and natural habitats. Furthermore, we tested the effect of the “sowing season” (autumn and spring) on seedling emergence and survival. Overall, seedling establishment of both species was enhanced by light and water. However, we found important interactions among climate, habitat, and disturbance on both species’ emergence and survival. The differences between habitats were more evident in the mild-summer climate than in the warm-summer climate. Our results also suggested that seedling survival may be enhanced by shrub cover in drier conditions (warm-summer climate). Eucalyptus globulus appears more sensitive to drought and disturbance changes than P. pinaster. In shrublands and mild-summer climate conditions, disturbance especially promoted E. globulus seedling establishment, while the forest canopy and the shade appeared to control it in both climatic conditions. After the first summer life, very low seedling survival was observed in both species, although the colonization of new areas appeared to be more limited for E. globulus. Our study suggests that climate conditions influence the effect (direction and intensity) of habitat and disturbance (plant–plant interactions) on seedling survival. Thus, the effect of light availability (forest canopy) and disturbance (vegetation removal) on these species establishment is climate context-dependent. This study presents very useful information to understand future shifts in these species distribution and has direct applications for the management of natural establishment outside the planted areas, and the management of the understorey to favor forest regeneration or limit forest colonization.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

Enhancement of polysaccharides production in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> by the addition of ethyl acetate extracts from <Emphasis Type="Italic">Eupolyphaga sinensis</Emphasis> and <Emphasis Type="Italic">Catharsius molossus</Emphasis>

To screen stimulators from Chinese medicinal insects for mycelial growth and polysaccharides production of Ganoderma lucidum, G. lucidum was inoculated into the media with and without supplementation of medicinal insect extracts. The ethyl acetate extract of Eupolyphaga sinensis at 55 mg l⁻¹ lead to significant increase in both biomass and intracellular polysaccharides (IPS) concentration from 8.53 ± 0.41 to 14.16 ± 0.43 and 1.28 ± 0.09 to 2.13 ± 0.11 g l⁻¹, respectively. In addition, the ethyl acetate extract of Catharsius molossus at 55 mg l⁻¹ significantly enhanced extracellular polysaccharides (EPS) production; the EPS yield increased from 350.9 ± 14.1 to 475.1 ± 15.3 mg l⁻¹. There were no new components in the two types of polysaccharides obtained by the addition of the insect extracts.     

<Emphasis Type="Italic">glyA</Emphasis> gene knock-out in <Emphasis Type="Italic">Escherichia coli</Emphasis> enhances L-serine production without glycine addition

In E. coli, glyA encodes for serine hydroxymethyltransferase (SHMT), which converts L-serine to glycine. When engineering L-serine-producing strains, it is therefore favorable to inactivate glyA to prevent L-serine degradation. However, most glyA knockout strains exhibit slow cell growth because of the resulting lack of glycine and C₁ units. To overcome this problem, we overexpressed the gcvTHP genes of the glycine cleavage system (GCV), to increase the C₁ supply before glyA was knocked out. Subsequently, the kbl and tdh genes were overexpressed to provide additional glycine via the L-threonine degradation pathway, thus restoring normal cell growth independent of glycine addition. Finally, the plasmid pPK10 was introduced to overexpress pgk, serA ^Δ197 , serC and serB, and the resulting strain E4G2 (pPK10) accumulated 266.3 mg/L of L-serine in a semi-defined medium without adding glycine, which was 3.18-fold higher than the production achieved by the control strain E3 (pPK10). This strategy can accordingly be applied to disrupt the L-serine degradation pathway in industrial production strains without causing negative side-effects, ultimately making L-serine production more efficient.     

Organogenesis and <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Eucalyptus saligna</Emphasis> with <Emphasis Type="Italic">P5CS</Emphasis> gene

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ¹-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied. The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots was similar in both types of plants.