Effect of Light and Moisture Conditions and Seed Age on Germination of Three Closely Related Myosotis Species

Germination of three closely related species from the Myosotis palustris group (M. nemorosa, M. palustris subsp. laxiflora, M. caespitosa) differing in their habitats and capacity for clonal growth, was compared in two greenhouse experiments. To evaluate both inter- and intraspecific variation, each species was represented by seeds from several populations. Final germination percentage and germination rates T₅₀ were compared both between species and populations within species. In the first experiment, we studied the influence of two external factors, moisture and light. Four moisture levels (dry, wet, periodically flooded and permanently flooded soil) and three types of shading (without shading, shaded with green foil, shaded with solid paper sheet) were combined in a complete factorial design. In all three species, total germination percentage was the same in the three wettest treatments, and decreased in the dry treatment. Germination in the treatments shaded with green foil (simulating vegetation cover, which changed light quality) was significantly slower than in treatments without shading and treatments shaded with a solid paper sheet. There were significant differences among species, but we also found very pronounced differences among populations within a species. M. caespitosa had the uniformly highest germination percentage (reaching in some cases 100%) and also fastest germination. Germination of M. palustris subsp. laxiflora populations was slower and reached lower final proportions, and medium variability among populations. Inter-population variability in the final germination percentage was highest, and the final germination the lowest in M. nemorosa. In addition, M. nemorosa, a species typical for permanent meadow communities was delayed by permanent flooding. In the second experiment, we studied the effects of seed age and storage conditions. Three combinations of seed age and storage were used: younger seeds (half year old) with no-chilling, younger seeds with chilling and old seeds (three years old) with chilling. M. caespitosa had again the highest final germination percentage and fastest germination rates T₅₀. In addition, final germination percentage of this species slightly increased with seed age, whereas it decreased in the other two species. The germination behaviour corresponded well to expectation based on species life histories and habitat preferences. Remarkably stable and high germination percentages and fastest germination rates T₅₀ were ascertained in M. caespitosa, a species of disturbed habitats, with lowest capacity for clonal spread. M. palustris subsp. laxiflora (species with highest clonal capacity) and M. nemorosa (species with medium clonal potential) achieved lower, but still very high final germination percentage. In addition, M. nemorosa showed the highest inter-populations variability in our experiments.     

Flow cytometric and karyological analyses of Calendula species from Iberian Peninsula

Calendula L. (Asteraceae) is a taxonomically and cytologically complex genus due to its high morphological and karyological variation. To gather consistent cytological information aiming to consolidate the existing knowledge, sustain the taxonomic revision of the genus and explore the evolutionary relationships among species, the genome size and chromosome number of the Iberian Peninsula representatives of this genus were assessed. The study included 11 taxa that occur in the Iberian Peninsula, one in Madeira and two from Morocco. Chromosome counts were made using the squash technique in root tips and flower buds, while nuclear DNA contents were assessed using propidium iodide flow cytometry. The following chromosome numbers are reported: 2n = 44 for C. arvensis, 2n = 30 for C. tripterocarpa, and 2n = 32 for the remaining Iberian taxa. The genome size of Calendula species was assessed for the first time and ranged from 1.75 pg/2C in C. maroccana to 5.41 pg/2C in C. arvensis. Within the complex formed by C. incana and C. suffruticosa, a gradient of genome size values was obtained. Intraspecific variation in genome size was detected in some taxa. The obtained genome size values and their variation are discussed in the light of the theories proposed for the speciation of the genus, with events of hybridization, genome duplication and dysploidy being hypothesized to play a major role in the evolution of this genus.     

Correlation between the production of exopolysaccharides and oxalic acid secretion by Ganoderma applanatum and Tyromyces palustris

The secretion of exopolysaccharides and oxalic acid in cultures of a white rot Ganoderma applanatum strain and a brown rot Tyromyces palustris strain were tested in terms of culture time, pH range, and temperature. The high yield of exopolysaccharides (EPS) required a moderate temperature of 28 °C for G. applanatum and 20 °C for T. palustris. G. applanatum and T. palustris accumulated more EPS when the concentration of the carbon source (maltose for G. applanatum and fructose for T. palustris) was 30 g/L. The results indicate that the production of oxalic acid by G. applanatum is correlated with the initial pH value of the culture medium and the concentration of oxalic acid increased to 1.66 ± 0.2 mM at the initial pH of 6.5 during the fungal growth. During the growth of T. palustris, the reduction of the initial pH value of the growing medium lowered the oxalic acid concentration from 7.7 ± 0.6 mM at pH 6.0 to 1.99 ± 0.2 mM at pH 3.5. T. palustris accumulated considerably more oxalic acid than G. applanatum and its presence did not affect significantly the production of exopolysaccharides. We also observed that the maximum amounts of exopolysaccharides secreted during cultivation of G. applanatum and T. palustris were 45.8 ± 1.2 and 19.1 ± 1.2 g/L, respectively.     

Plant regeneration from the embryogenic calli of five major Miscanthus species, the non-food biomass crops

With the growing shortage of oil, coal, and other traditional fossil fuels, scientists in various fields have been looking for new fuel sources to solve the energy crisis. The genus Miscanthus is an ideal biofuel crop due to its rapid vegetative growth and its potential for high biomass yields. Plant regeneration through somatic embryogenesis is a viable method to achieve large-scale production of plant biomass. Callus induction from immature inflorescences of five Miscanthus species was performed on two different media, and the relative rates of callus proliferation were calculated. The highest multiplication coefficient, 3.92, was obtained with M. sacchariflorus ssp. lutarioriparius when the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the medium was 4.0 mg/L, and this species also performed the best at the induction phase. The proliferation coefficient for M. floridulus increased to 3.19 when the concentration of 2,4-D was decreased from 4.0 to 2.0 mg/L. When the concentration of 2,4-D was 2.0 mg/L, the proliferation coefficient for M. sinensis was 2.47. In M. sacchariflorus, the proliferation coefficients were 2.89 and 2.49 for the lower and higher concentrations of 2,4-D, respectively. The multiplication coefficient of M. x giganteus was 2.60 on medium containing 4.0 mg/L 2,4-D. Three different regeneration media were tested to induce shoots in vitro. M. floridulus and M. sacchariflorus regenerated shoots at 100% frequency in three different regeneration media. The regeneration rate for M. sacchariflorus ssp. lutarioriparius reached 99.0% on medium containing 4.0 mg/L?N ⁶ -Benzylaminopurine (6-BA) and 0.1 mg/L α-Naphthaleneacetic acid (NAA). The best regeneration rate of M. sinensis was 35.2% using 2.0 mg/L 6-BA and 0.3 mg/L NAA, whereas the regeneration rate of M. x giganteus was 57.4% on medium supplemented with 3.0 mg/L 6-BA and 0.2 mg/L NAA. The in vitro-derived plantlets of all five species had 100% rooting rates on basal MS medium without supplementation. The survival rates of plantlets were above 90% for each species when subsequently grown outdoors.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of endangered <Emphasis Type="Italic">Mammillaria</Emphasis> genus (Cactaceae) species and genetic stability assessment using SSR markers

In vitro propagation protocols were established for endangered species of cacti Mammillaria hernandezii, M. dixanthocentron, and M. lanata. In vitro-germinated seedlings were used as the explant source. Three explant types were evaluated as apical, basal, and lateral stem sections. Shoot multiplication was achieved using Murashige and Skoog (MS) medium supplemented with benzyladenine, kinetin, meta-topolin, and thidiazuron in equimolar concentrations (0.0, 0.4, 1.1, 2.2, 4.4, and 8.9 μM). Shoot regeneration was obtained primarily in the lateral stem section explants. In M. hernandezii, an average of 7.4 shoots was regenerated in MS medium with 2.2 μM meta-topolin. M. dixanthocentron and M. lanata averaged 16.7 and 17.9 shoots/explant, respectively, in MS medium supplemented with 1.1 μM meta-topolin. Rooting occurred in MS medium without growth regulators. Three in vitro culture cycles were performed to validate the propagation protocols and to verify genetic stability. Shoots were collected in each cycle and genomic DNA was extracted. Amplified microsatellites were used to compare each genotype with its respective donor plant. Polymorphic information content analysis showed low levels of intra-clonal polymorphisms—M. hernandezii 0.04 and M. dixanthocentron and M. lanata both 0.12. More than 95% of the plants were successfully acclimatized in the greenhouse. After 12 months, plants of M. hernandezii reached the flowering stage; M. dixanthocentron and M. lanata flowered at 24 mo.     

Nontuberculous mycobacteria: an emerging risk in engineered environmental habitats

Nontuberculous mycobacteria (NTM) are microorganisms commonly living in the environment. Nevertheless, most of them are opportunistic pathogens. To verify concentrations of NTM in some man-made habitats, analyses were performed on water and surface samples, and the data were correlated to the global microbiological quality of water. Most of the drinking water samples (98 %) complied with the microbial requirements established by the European Directive 98/83/EC on drinking water when Escherichia coli was considered. Low counts of heterotrophs were also obtained. NTM were isolated from 72 % of the analyzed samples. Tap water from private buildings, schools and hospitals provided positive results for NTM, with comparable densities ranging from 1 to 6?×10² CFU/L. NTM were also found in swimming pool water samples, with concentrations ranging from 29 to 3?×10⁴ CFU/L, as well as in 70 % of the surface sample. The most frequently isolated species were Mycobacterium mucogenicum, M. intracellulare and M. terrae. These yields confirm that no correlation exists between the monitoring controls carried out by law and the occurrence of these bacteria that may represent a potential risk, especially for immunocompromised people and vulnerable groups.     

A natural plant growth promoter calliterpenone from a plant Callicarpa macrophylla Vahl improves the plant growth promoting effects of plant growth promoting rhizobacteria (PGPRs)

Experiments were conducted to evaluate the efficacy of calliterpenone, a natural plant growth promoter from a shrub Callicarpa macrophylla Vahl., in enhancing the growth and yield promoting effects of plant growth promoting rhizobacteria (PGPRs), in menthol mint (Mentha arvensis L).This study is based on our previous results indicating the microbial growth promotion by calliterpenone and assumption that application of calliterpenone along with PGPRs will improve the population of PGPRs resulting in higher impacts on plant growth and yield. Of the 15 PGPRs (identified as potent ones in our laboratory), 25 μl of 0.01 mM calliterpenone (8.0 μg/100 ml) was found to be useful in improving the population of nine PGPRs in culture media. The five selected strains of PGPRs exhibiting synergy with calliterpenone in enhancing growth of maize compared to PGPR or calliterpenone alone were selected and tested on two cultivars (cvs. Kosi and Kushal) of M. arvensis. Of the five strains, Bacillus subtilis P-20 (16S rDNA sequence homologous to Accession No NR027552) and B. subtilis Daz-26 (16SrDNA sequence homologuos to Accession No GU998816) were found to be highly effective in improving the herb and essential oil yield in the cultivars Kushal and Kosi respectively when co-treated with calliterpenone. The results open up the possibilities of using a natural growth promoter along with PGPRs as a bio-agri input for sustainable and organic agriculture.     

Main rhizosphere characteristics of the Cd hyperaccumulator Rorippa globosa (Turcz.) Thell

Aim

This article was aimed to explore the main rhizospherial properties of the Cd hyperaccumulator R. globosa compared to those of the non hyperaccumulator Rorippa palustris (Leyss.) Bess. representing the same genus (Rorippa) of Cruciferae.

Method

Pot culture experiments using soil spiked with Cd as CdCl₂·2.5H₂O and rhizobags were conducted to determine the differences in Cd accumulation vs. pH, dissolved organic carbon (DOC), Cd chemical fractionation, enzyme activities, and microorganism number in the rhizospheres of R. globosa and R. palustris, and in the bulk soils.

Results

Experiments on Cd uptake by R. globosa and R. palustris from soil spiked with different doses of Cd ranging from 0 to 40 mg?kg^?1, confirmed Cd-hyperaccumulating properties of R. globosa (Cd accumulation in the above-ground organs >100 mg kg^?1, enrichment factor EF> 1, translocation factor TF> 1, no significant biomass reduction at Cd doses >10 mg kg^?1) and the lack of such properties in R. palustris, which made these species suitable for comparative studies. The pH value was found to be a constant, specific property of the rhizosphere of R. globosa and R. palustris, and of the bulk soil, independent on the Cd dose, however the differences were rather small: by 0.2 unit lower in the rhizosphere of R. globosa, and only by 0.1 unit lower in the rhizosphere of R.. palustris compared to the bulk soil. Chemical fractionation of Cd, i.e. its affinity to pools of different binding strength, also appeared to be a specific feature of a rhizosphere and soil independent on the Cd dose. It exhibited a unique capability of the rhizosphere of the Cd-hyperaccumulator R. globosa to mobilize Cd, which enriched the most labile exchangeable fraction in 24.4 % and the immobile residual fraction in 42.3 %, compared to 19.3 % and 50.8 % in the bulk soil and in the rhizosphere of the non-hiperaccumulator R.palustris that did not show significant difference (p?<?0.05) from the bulk soil. In turn, DOC concentrations, enzymatic (urease and catalase) activity and microorganism (bacteria, fungi and actinomycetes) growth in rhizosphere soils were largely influenced by different Cd doses, although they were always considerably higher in the rhizosphere soils of R globosa, than in the rhizosphere of R. palustris and in the bulk soil, in particular at Cd doses ≥10 mg kg^?1.

Conclusion

pH and DOC changes in the rhizosphere of the Cd-hyperaccumulator R. globosa were found to be of a minor importance. The alteration of Cd chemical fractionation consisting in substantial reduction of the immobile residual pool and Cd enrichment primarily in the most labile exchangeable fraction, along with over 2-fold higher number of microorganisms was considered to be the driving force of Cd hyperaccumulation.     

Changes in aroma profiles of 11 Indian Ocimum taxa during plant ontogeny

The genus Ocimum is an important source of many essential oils and aromatic chemicals used principally in the food and cosmetic industries. The aromas (compositions) of essential oils of genus Ocimum are characterized by a great chemical variability affecting the commercial value of this genus. This study was conducted to evaluate the ontogenic variations in aroma profiles of 11 Indian Ocimum taxa belonging to 5 Ocimum species, viz. O. basilicum L., O. americanum L., O. gratissimum L., O. tenuiflorum L., and O. kilimandscharicum Guerke, grown in foot hills of Uttarakhand, India during rain-autumn cropping season. Essential oil yield was found to vary significantly during different phenophases: from 0.34 to 0.87 % in four landraces of O. basilicum, 0.16 to 0.38 % in ‘Green and Purple’ of O. tenuiflorum, 0.23 to 0.43 % in O. americanum, 0.34 to 0.78 % in O. kilimandscharicum and 0.48 to 0.68 % in O. gratissimum. The variation in composition of the essential oils was analyzed by gas chromatography with flame ionization detection and gas chromatography with mass spectrometry. Analyses of the essential oils led to the identification of 95 constituents forming 93.8–99.7 % of the total essential oil compositions, with phenyl propanoids (≤0.1–90.0 %) and monoterpenoids (2.8–95.5 %) as the prevalent components. Substantial variations in essential oil yield, and qualitative and quantitative compositions due to different stages of plants growth were noticed. These results indicated that the knowledge of appropriate growth stage is very important to obtain higher essential oil yield and desired active constituents of Ocimum, which were used extensively in food, cosmetics and allied industries.     

Salicylic acid affects growth,essential oil and chemical compositions of thyme (Thymus daenensis Celak.) under reduced irrigation

Salicylic acid (SA) may reduce the negative impact of water deficit on growth and metabolite yield of Thymus daenensis Celak subsp. daenensis Celak. The effect of foliar application of SA and reduced irrigation on growth, oil yield, chemical components, and antibacterial and antioxidant activities of T. daenensis in field condition were investigated. Treatments comprised 0.0, 1.5 and 3.0 M SA applied to plants under normal irrigation and stressed conditions. Results indicated that irrigation regime had a significant effect on growing degree days (GDD) required to reach early and full flowering. Foliar application of SA influenced GDD from early growing stage to 50 % and full flowering, minimum radius and canopy diameter. The highest values of oil content (3.2 % v/w) and yield (14.9 g m^?2) were obtained from application of 3.0 M SA. Percentage of some chemical constituents in the essential oil extracted from the plants under stress was higher than non-stressed plants. Thymol content was significantly reduced under stressed conditions. Foliar application of SA significantly improved carvacrol, α-thujene, α-pinene and p-cymene contents in the oils, but reduced thymol and, β-caryophyllene amounts. Our results showed that foliar application of SA reduced the negative effect of water deficit on thymol content in the essential oil of T. daenensis. The essential oils of T. daenensis exhibited antioxidant and antibacterial activities when plants were sprayed with 1.5 and 3.0 M SA, respectively.     

Fungal treatment followed by FeCl3 treatment to enhance enzymatic hydrolysis of poplar wood for high sugar yields

Fungal treatment followed by FeCl₃ treatment was used to improve saccharification of wood from Populus tomentosa. Combined treatments accumulated lignin and slightly degraded cellulose, whereas almost all hemicelluloses were removed. The white rot fungus, Trametes orientalis, and the brown rot fungus, Fomitopsis palustris, both accompanied by FeCl₃ post-treatment resulted in 98.8 and 99.7 % of hemicelluloses loss at 180 °C, respectively, which were over twice than that of hot water pretreatment at the same level. In addition, the solid residue from the T. orientalis-assisted and F. palustris-assisted FeCl₃ treatment at 180 °C released 84.5 and 95.4 % of reducing sugars, respectively: 1.4- and 1.6-fold higher than that of FeCl₃ treatment alone at the same temperature. Combined treatments disrupted the intact cell structure and increased accessible surface area of cellulose therefore enhancing the enzymatic digestibility, as evidenced by XRD and SEM analysis data.     

Essential oils of Bidens tripartita L. collected during period of 3 years composition variation analysis

The aim of this study was to evaluate the variation of essential oils composition of Bidens tripartita L. collected during three successive years (2009–2011). Essential oils were obtained by supercritical CO₂ extraction, applying gas chromatographic–mass spectrometric (GC–MS) analysis for identification of volatile compounds afterwards. The essential oils of B. tripartita showed a characteristic chemical composition from year to year, observing both quantitative and qualitative compounds differences. The yield of essential oils was 22 and 35 % higher in 2010 year material than in 2009 and 2011 year, respectively. The main compounds found in the B. tripartita essential oils were α-pinene (3.7–12.1 %), p-cymene (2.8–8.0 %), β-ocimene (40.5–45.9 %), β-elemene (9.9–15.6 %), iso and α-caryophyllenes (4.3–6.8 % and 5.2–8.2 %), and α-bergamotene (3.3–9.4 %). To determine the significance of changes in the identified compounds in the samples, representing different plant collection year, statistical hypothesis testing was applied. For classification of these samples to the groups and evaluation of similarity between them principal component analysis, multivariate analysis of variance and hierarchical cluster analysis techniques were used. The correlation analysis helped to find out the strength of linear relationship between amount of each compound and meteorological data (temperature and precipitations).     

The Effect of Some Natural Essential Oils Against Bovine Mastitis Caused by <Emphasis Type="Italic">Prototheca zopfii</Emphasis> Isolates In Vitro

The aim of the study was to evaluate the effect of essential oils obtained from Thymus vulgaris L., Origanum vulgare L., Origanum majerana L., Mentha × piperita L. and Allium ursinum L. against Prototheca zopfii strains that cause inflammation of the udder (mastitis) in cows. The study was conducted on ten strains derived from milk samples. The microdilution method was used to determine the sensitivity of P. zopfii strains to the studied essential oils, and the disk diffusion method was used to determine the sensitivity to antifungal chemotherapeutics. The plates were incubated for 48 h at 37 °C under aerobic conditions. All strains of algae were sensitive to the essential oils marjoram, thyme and oregano and resistant to mint and garlic oils. MIC values ranged from 0.25 to 1 μl/ml. Marjoram oil demonstrated the greatest activity, and oregano oil the weakest. Among the antifungal agents tested, 90% of strains showed sensitivity to nystatin. One of the tested strains (71/IV) was resistant to all investigated antifungal agents. The tested essential oils are known to have anti-algae activity and can be used as natural agents for prophylaxis in animals, particularly in mastitis-affected cows.     

Comparative Genome Analysis of Fish and Human Isolates of Mycobacterium marinum

Mycobacterium marinum is a major causative agent of mycobacteriosis in fish that has a broad range of hosts, including in human isolates. So far, genomic analyses have focused on the human isolate. Here, we compared the draft genome sequences of two strains of M. marinum isolated from fish (MB2 and Europe) with the M. marinum M isolated from humans. M. marinum MB2 and Europe have single, circular chromosomes of 6,134,389 and 6,029,340 bp, and average G + C contents of 65.7 and 65.5 %, respectively. A total of 5,464 coding DNA sequences were annotated in both M. marinum MB2 and Europe genome. Dot plot analyses showed that M. marinum MB2 and Europe were closer to M. marinum M when compared to three other Mycobacterium species. The insertion/deletion gene analysis showed that M. marinum MB2 and Europe contained 342 and 487 genes that were not found in M. marinum M, and lacked 625 and 776 genes found in M. marinum M, respectively. Most of the inserted and deleted genes were classified in the fatty acid, lipid, and isoprenoid subsystem and the virulence, disease, and defense subsystem. Therefore, these results provide insights into the genomic diversity associated with variable hosts and pathogens.     

Chemical Diversity of Volatile Compounds of Mints from Southern Part of Pannonian Plain and Balkan Peninsula – New Data

Mints are the most popular economic and traditional herbs. The aim of this article was chemical characterization of volatile compounds from wild populations of Mentha aquatica, M. arvensis, M. longifolia, M. microphylla, M. pulegium, M. spicata, M.×dumetorum, M.×gentillis and M.×verticillata, as well as cultivated samples of M. spicata, M.×piperita ‘Alba’ and M.×piperita ‘Crispa’. Analyses were performed directly from dried aerial parts (herb) of collected samples by headspace gas chromatography‐mass spectrometry. In total 54 compounds were detected, representing from 89.99 % to 99.66 % of volatile fractions of all investigated samples. The recorded volatiles were primarily monoterpene hydrocarbons and oxygenated monoterpenes, while oxygenated aromatic monoterpenes, sesquiterpene hydrocarbons and aliphatic compounds were present in lower concentrations in analyzed samples. The major components were linalool, limonene, 1,8‐cineol, α‐terpinyl acetate, pulegone, β‐pinene and menthol. The cluster analysis revealed five main groups or chemotypes according to qualitative and quantitative content of volatiles, as well as similarities among samples. These results contribute to the knowledge on the mints chemistry in Pannonian Plain and Balkan Peninsula.     

Antioxidant activities of the volatile oils and methanol extracts from olive stems

This study was designed to examine the chemical composition and antioxidant activities of the volatile oils and methanol extracts of Olea europaea L. (cvs) chemlali and neb jmel stems. GC and GC–MS analyses of the volatile oils resulted in the identification of 38 and 35 compounds, representing 91.1 and 87.4 % of the volatile oils. Phenylethyl alcohol was found in the volatile oil of each cultivar, which was also the major volatile component of cv. chemlali and cv. neb jmel stems. Besides benzyl alcohol, methyl salicylate and 3-ethenylpyridine were the main volatile compounds of cv. chemlali, while nonanal, 3-ethenylpyridine and benzyl alcohol of cv. neb jmel stems were also the main constituents. Significant differences were also found in total tannin contents among two cultivars, representing 8.10 mg CEQ/g DW in cv. chemlali and 20.47 mg CEQ/g DW in cv. neb jmel. The highest contents of total phenols and o-diphenols were observed in stems extracts of cv. neb jmel (78.26, and 9.56 mg/100 g, respectively). The HPLC profiles for methanol extracts from stems of cv. chemlali and cv. neb jmel showed that oleuropein, vanillic acid and gallic acid were the predominant free phenolic compounds. Antioxidant activities of the volatile oils and the methanolic extract from stems parts were evaluated by DPPH and ABTS⁺ radical-scavenging activity assays. In all tests, methanolic extracts obtained from stems parts showed better antioxidant activity than volatile oils. Principal components analysis of the phenolics content and antioxidant activities showed discrimination between methanol extracts of the two cultivars.     

Use of Rhodopseudomonas palustris P1 stimulated growth by fermented pineapple extract to treat latex rubber sheet wastewater to obtain single cell protein

Latex rubber sheet wastewater (non sterile wastewater: RAW) was treated efficiently using a stimulated Rhodopseudomonas palustris P1 inoculum with added fermented pineapple extract (FPE) under microaerobic light conditions. Optimization of wastewater treatment conditions using a central composite design (CCD) found that a 3 % stimulated P1 inoculum with 0.9 % added FPE and a 4-day retention time (RT) were the most suitable conditions. Calculations from CCD experiments predicted that a chemical oxygen demand (COD) of 3,005 mg/L could be 98 % removed, together with 79 % of suspended solids (SS) and 72 % of total sulfide (TtS). No H₂S was detected, production costs were low and single cell protein (SCP) was a by-product. The results of the verification test had an error of only 4–8 % and confirmed removal of COD (initial COD 2,742 mg/L), SS and TtS at 94 %, 75 % and 66 %, respectively. These values were less than the best set obtained from the CCD experiment (2 % stimulated P1 inoculum, 0.75 % FPE and 4 days RT); upon repeating, this set could reduce 96 % of the COD, 78 % SS and 71 % TtS. The treated wastewater met the standard guidelines for irrigation use and no H₂S was detected. The biomass obtaining after wastewater treatment from the best set consisted mostly of R. palustris P1; the biomass of this set had 65 % protein, 3 % fat, 8 % carbohydrate, 14 % ash and 10 % moisture. The results demonstrated that an inoculum of stimulated P1 grew well in RAW supplemented with FPE and could be considered to be an appropriate technology for effectively treating wastewater, with SCP as a by-product.     

Endophytic fungi associated with Macrosolen tricolor and its host Camellia oleifera

Endophytic fungi play an important role in terrestrial ecosystem, while little is known about those in hemi-parasitic plants, a group of special plants which absorb nutrients from its hosts by haustoria. The relationship of the endophytes in the two parts of the bipartite systems (hemiparasites together with their hosts) is also poorly understood. Endophytic fungi of a hemi-parasitic plant Macrosolen tricolor, and its host plant Camellia oleifera were investigated and compared in this study. M. tricolor contained rich and diversified endophytic fungi (H′ = 2.829), which consisted mainly of ascomycetes, distributed in more than ten orders of four classes (Sordariomycetes, Dothideomycetes, Leotiomycetes and Eurotiomycetes) besides Incertae sedis strains (23.2 % of total). In addition, 2.2 % of isolates were identified to be Basidiomycota, all of which belonged to Agaricomycetes. Obvious differences were observed between the endophytic fungal assembles in the leaves and those in the branches of M. tricolor. The endophytic fungi isolated from C. oleifera distributed in nearly same orders of the four classes of Ascomycota and one class (Agaricomycetes) of Basidiomycota as those from M. tricolor with similar proportion. For both M. tricolor and C. oleifera, Valsa sp. was the dominant endophyte species in the leaves, Torula sp. 1 and Fusarium sp. 1 were the dominant endophytic fungi in the branches. The similarity coefficient of the endophyte assembles in the two host was 64.4 %. Canonical correspondence analysis showed that the endophyte assembles of M. tricolor and C. oleifera were significantly different (p < 0.01).     

Characterization and distinction of two subspecies of Eryngium duriaei J. Gay ex Boiss., an Iberian endemic Apiaceae, using flow cytometry and essential oils composition

Eryngium duriaei (Apiaceae) is an endemic taxa from Iberian Peninsula. Some doubts exist in the literature about the taxa relationships, especially among Iberian populations at different altitudes. Also, as other Apiaceae, this species presents a large potential for essential oil production. Considering all this, a multidisciplinary study comprising taxonomic, cytological (using flow cytometry and chromosome counts) and chemical (essential oils) analyses was performed with the objective to morphologically characterize this species and to evaluate the cytotaxonomical and chemical diversity of E. duriaei Portuguese populations. FCM and chromosome counts have shown that every individual presented the same ploidy level, i.e., 2n = 2x = 16 chromosomes. However, flow cytometric analyses revealed that individuals of E. duriaei from higher altitudes (»1,700 m) presented a significantly higher genome size than those belonging to E. duriaei populations below 1,700 m (2C = 6.20 ± 0.04 vs. 2C = 5.52 ± 0.05 pg). Moreover, the essential oils analyses revealed that most chemical constituents were sesquiterpenes, but relevant differences in the main components were observed: α-neocallitropsene (28–53 %), β-betulenal (8.5–15.8 %) and 14-hydroxy-β-caryophyllene (5.8–13.7 %) were the main compounds of Eryngium duriaei oil below 1,700 m, whereas caryophyllene oxide (47 %) and E-caryophyllene (6 %) were the major compounds of E. duriaei oil of higher altitude populations. The results provide important evidences to support the taxonomic separation of E. duriaei in two taxa: E. duriaei J. Gay ex Boiss. subsp. duriaei and E. duriaei subsp. juresianum (M. Laínz) M. Laínz, as previously considered by this author.