Abstract

Biological nitrogen fixation by rhizobia-nodulated legumes reduces the dependence on synthetic nitrogen fertilizers. Identification of locally adapted rhizobia may uncover economically valuable strains for sustainable agriculture. This study investigated the diversity and symbiotic potential of rhizobia associated with three Medicago species from Eastern Romania's ecosystems. Phenotypic screening ensured that only rhizobial species were retained for molecular characterization. 16S rDNA sequencing clustered the isolates into four distinct groups: Sinorhizobium meliloti, Sinorhizobium medicae, Rhizobium leguminosarum, and Mesorhizobium spp. The chromosomal genes (atpD, glnII, recA) and nifH phylogenies were congruent, while the nodA phylogeny grouped the Mesorhizobium spp. isolates with R. leguminosarum. Medicago sativa was the most sampled plant species, and only S. meliloti and R. leguminosarum were found in its nodules, while Medicago falcata nodules hosted S. meliloti and Mesorhizobium spp. Medicago lupulina was the only species that hosted all four identified rhizobial groups, including S. medicae. This study provides the first report on the Mesorhizobium spp. associated with M. falcata nodules. Additionally, R. leguminosarum and two Mesorhizobium genospecies were identified as novel symbionts for Medicago spp. Comparative analysis of Medicago-associated rhizobia from other studies revealed that differences in 16S rDNA sequence type composition were influenced by Medicago species identity rather than geographic region.

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This research is focused on cellular response to sodium metabisulphite (E223), sodium benzoate (E211), and sodium nitrite (E250) application on two normal mammalian cells, namely MCF-12A (ATCC CRL-3598) and Vero (ATCC CCL-81). The monitored parameters were cell viability (MTT assay), cell morphology (optical microscopy) and cell survival (clonogenic assay). The treatment was applied in doses of 12.5; 25; 50 and 100 µg/mL. According to all tests assessed, our results proved a dose-response relationship, the most sensibility presented the MCF-12A cell line.

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The study aimed to evaluate the effects of non-thermal plasma activated water (PAW) in combination with low temperature (15 degrees C) on the biosynthetic potential, antioxidant activity and enzymes activity in wheat (Triticum aestivum L. cv. Glosa) sprouts. The exposure to three different types of PAW (PAW1, PAW2 or PAW3) and low temperature (15 degrees C) had positive influence on wheat sprouts. Content in proteins and photosynthetic pigments and activity of superoxide dismutase increased in wheat sprouts at day 3 and shoots at day 8. Peroxidase was stimulated in wheat sprouts (day 3) while catalase activation was detected in shoots (day 8). Polyphenol oxidase, responsible for enzymatic browning, was inhibited in shoots at day 8. PAW1 boosted free phenolic content in shoots at day 8. An increment in the antioxidant activity of bound phenolic fraction of shoots (day 8) under treatment with PAW1 was also detected. Overall, non-thermal PAW in combination with low temperature (15 degrees C) is an efficient approach to enhance the quality of wheat (Triticum aestivum L. cv. Glosa) sprouts.

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The present study aimed to investigate the effects of non-thermal plasma-activated water (PAW) on Triticum aestivum L. cv. Glosa sprouts. Two types of PAW were generated by exposing distilled water to a high-voltage electric discharge. Wheat caryopses were treated either with PAW1 (25 mg/L NO3-, 4 mg/L NO2-, and 6 mg/L H2O2) or PAW2 (35 mg/L NO3-, 5 mg/L NO2-, and 7.5 mg/L H2O2) for 8 days, with samples being collected at days 1, 2, 3, and 8. The germination rate, growth parameters, protein, photosynthetic pigments, total phenolic contents, antioxidant activity of free and bound phenolic fractions, and activity of antioxidant enzymes were evaluated. Both PAW1 and PAW2 had positive effects on Triticum aestivum L. cv. Glosa sprouts. PAW2 had a better impact on sprouts with respect to growth parameters, free phenolic content, and antioxidant activity of the free phenolic fraction (day 3), the accumulation of proteins, and photosynthetic pigments (day 8). In conclusion, exposure to PAW increases the quality of Triticum aestivum L. cv. Glosa sprouts. Further optimization of PAW treatment is required for the most favorable impact on the accumulation of biomass and health-promoting compounds in sprouts.

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The Artemisia genus includes a large number of species with worldwide distribution and diverse chemical composition. The secondary metabolites of Artemisia species have numerous applications in the health, cosmetics, and food sectors. Moreover, many compounds of this genus are known for their antimicrobial, insecticidal, parasiticidal, and phytotoxic properties, which recommend them as possible biological control agents against plant pests. This paper aims to evaluate the latest available information related to the pesticidal properties of Artemisia compounds and extracts and their potential use in crop protection. Another aspect discussed in this review is the use of nanotechnology as a valuable trend for obtaining pesticides. Nanoparticles, nanoemulsions, and nanocapsules represent a more efficient method of biopesticide delivery with increased stability and potency, reduced toxicity, and extended duration of action. Given the negative impact of synthetic pesticides on human health and on the environment, Artemisia-derived biopesticides and their nanoformulations emerge as promising ecofriendly alternatives to pest management.

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Eight wild rose hip genotypes from different altitudes varying from 3m to 902m were analyzed in order to evaluate the total polyphenols, flavonoids content and the antioxidant activity. The Rosa spp.rosehips collected from the Northeastern and the Southeasternof Romanianwere, as follows: Rosa canina, R. caesia, R. corymbifera, R. micrantha, R. nitidula, R. rubiginosa, R. subcanina, and R. vosagiaca. In some genotypes, the level of flavonoids and polyphenol content increased with the increasing altitude while in other it was observed a decrease. Polyphenol content reached a maximum of 144.36 mg GAE/g DW in R. rubiginosa whereas the lowest content of 61.72 mg GAE/g DW was recorded in R. caesia. The highest polyphenol content was reached at altitude of 860 m. The amount of flavonoids content ranged between 7.32 mg CE/g DW and 19.45 CE/g DW in R. caesia and in R. nitidula, respectively. The radical scavenging capacities of Rosa genotypes extracts were not positively correlated with altitude, except the R. corymbifera extractswhere the antioxidant activity increased with the increase of altitude.

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Thymus species are important aromatic, medicinal and culinary plants with a significant economic value. This study evaluated for the first time the chemical composition and in vitro bioactivities of the essential oils (EOs) from five Moldavian Thymus species (T. vulgaris, T. x citriodorus, T. calcareus) and cultivars (T. vulgaris 'Faustini', T. citriodorus 'Aureus'). The main compounds in Thymus EOs were: thymol in T. vulgaris and T. calcareus EOs (55.44% and 55.45%, respectively), lavandulol in T. x citriodorus EO (54.27%), and geraniol in T. citriodorus 'Aureus' and T. vulgaris 'Faustini' EOs (60.31% and 31.45%, respectively). T. vulgaris and T. calcareus EOs showed the most potent antioxidant activities (EC50=0.003 mg/mL in ABTS radical cation scavenging assay) and exhibited significant inhibitory effects against aflatoxin-producing Aspergillus flavus fungus (MIC=0.25 mu L/mL). At doses that provided micromolar concentrations of thymol, T. vulgaris and T. calcareus EOs acted genoprotective at preventive and interventional levels against H2O2-induced genomic damage in V79 cells, the former being more active (6.21% and 5.52% vs. 25.13% and 7.26% tail DNA in pre- and post-treatment protocols, respectively). The genoprotective effects may be ascribed to antioxidant potential and, possibly, to stimulation of DNA repair processes. The Moldavian Thymus species are valuable resources of bioactive EOs for pharmaceutical and food industries (T. vulgaris, T.calcareus) but also for flavor industry and perfumery (T. x citriodorus, T. citriodorus 'Aureus', T. vulgaris 'Faustini').

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The aim of this study was to evaluate the performances of Pichia kudriavzevii CR-Y103 yeast strain for the decolorization, biodegradation, and detoxification of cationic dye C.I. Basic Blue 41, a toxic compound to aquatic life with long-lasting effects. Under optimized cultural conditions (10.0-gL(-1) glucose, 0.2-gL(-1) yeast extract, and 1.0-gL(-1) (NH4)(2)SO4), the yeast strain was able to decolorize 97.86% of BB41 (50mgL(-1)) at pH 6 within 4h of incubation at 30 degrees C under shaken conditions (12,238.00-gh(-1) average decolorization rate) and 100% within 12h. The UV-Vis spectral analysis, high-performance liquid chromatography (HPLC), and Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the complete decolorization and degradation of the BB41 dye by P. kudriavzevii CR-Y103. Also, other seven yeast strains, isolated from soil, as P. kudriavzevii (CR-Y108, CR-Y119, and CR-Y112), Candida tropicalis CR-Y128, Cyberlindnera saturnus CR-Y125, and Candida solani CR-Y124 have shown a promising decolorizing potential of azo-dye BB41 (99.89-76.09% decolorization). Phytotoxicity, cytotoxicity, and genotoxicity assays on Trifolium pratense and Triticum aestivum seedlings confirmed the high toxicity of BB41 dye (500ppm), with inhibition on germination rate (%), root and shoot elongation, decreasing of mitoxic index value (with 34.03% in T. pratense and 40.25% in T. aestivum), and increasing the frequency of chromosomal aberrations (6.87 times in T. pratense and 6.25 times in T. aestivum), compared to control. The same biomarkers indicated the nontoxic nature of the BB41 degraded metabolite (500ppm) obtained after P. kudriavzevii CR-Y103 treatment. Moreover, the healthy monkey kidney cells (Vero cells) had a low sensitivity to BB41 biodegraded products (250gmL(-1)) (MTT cell viability assay) and revealed minor DNA damage (comet assay) compared to BB41 dye treatment. These findings show that P. kudriavzevii could be used in eco-friendly bioremediation technologies, applicable for reducing the toxicity of basic azo-dyes containing wastewaters.

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In consideration of the hazards associated with the presence of the textile azo-dye and their biotransformation products in the environment, the goal of this work was to study bioremediation process by the yeast strain Pichia kudriavzevii CR-Y103 related to the ability to degrade and detoxify the sulfonated Reactive Orange 16 azo-dye. In experimental conditions, the optimal inoculum/dye concentration ratio required for complete decolorization (100%) of culture medium and biomass within 24 h has been 1 g L-1 yeast cell (dry weight)/50 mg L-1 Reactive Orange 16. In the presence of 400 mg L-1 of Reactive Orange 16 (RO16), 95% of the dye was removed after 72 h of incubation. Also, the yeast strain could decolorize other eight textile dyes (56.48-99.98% decolorization within 24 h). NADH-DCIP reductase and azo reductase activities were significantly increased (ca. 5.4 times and ca. 37 times, respectively) during the decolorization process. UV-VIS spectra, high-performance liquid chromatography (HPLC), and Fourier transform infrared spectroscopy (FTIR) analysis confirmed the presence of new biotransformation products in extracted metabolites, highlighting the partial biodegradation of the dye by the new yeast isolate. The phytotoxicity evaluation strongly supported the decreased toxicity of biodegraded products as minor inhibition on germination (%), root and shoots elongation of T. pratense L. and T. aestivum L. seedlings. Increasing of mitotic index value and decreasing the frequency of chromosomal aberrations in tested plant meristem cells treated with biodegraded products, compared with RO16 treatment (500 ppm), confirmed their slightly toxic nature. A cell viability assay also confirmed the reduced toxicity of biodegraded products on healthy monkey kidney cells (Vero cells).

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The identification and conservation of indigenous rhizobia associated with legume plants and their application as biofertilizers is becoming an agricultural worldwide priority. However, little is known about the genetic diversity and phylogeny of rhizobia in Romania. In the present study, the genetic diversity and population composition of Rhizobium leguminosarum symbiovar trifolii isolates from 12 clover plants populations located across two regions in Romania were analyzed. Red clover isolates were phenotypically evaluated and genotyped by sequencing 16S rRNA gene, 16S-23S intergenic spacer, three chromosomal genes (atpD, glnll and recA) and two plasmid genes (nifH and nodA). Multilocus sequence typing (MLST) analysis revealed that red clover plants are nodulated by a wide genetic diversity of R. leguminosarum symbiovar trifolii sequence types (STs), highly similar to the ones previously found in white clover. Rhizobial genetic variation was found mainly within the two clover populations for both chromosomal and plasmid types. Many STs appear to be unique for this region and the genetic composition of rhizobia differs significantly among the clover populations. Furthermore, our results showed that both soil pH and altitude contributed to plasmid sequence type composition while differences in chromosomal composition were affected by the altitude and were strongly correlated with distance. (C) 2018 Elsevier GmbH. All rights reserved.

Abstract

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.

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The application of commercial rhizobial inoculants to legume crops is proving to be an alternative to synthetic fertilizer use. The challenge for sustainable agriculture resides in the compatibility between crop, inoculants and environmental conditions. The evaluation of symbiotic efficiency and genetic diversity of indigenous rhizobial strains could lead to the development of better inoculants and increased crop production. The genetic variability of 32 wild indigenous rhizobial isolates was assessed by RAPD (Random Amplified Polymorphic DNA). The strains were isolated from red clover (Trifolium pratense L.) nodules from two distinct geographical regions of Northern and Eastern Romania. Three decamer primers were used to resolve the phylogenetic relationships between the investigated isolates. Cluster analysis revealed a high diversity; most strains clustered together based on their geographical location.

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The changes in superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, and in soluble protein level were analysed in Triticum aestivum cv Beti seedlings, after seed exposure to copper, provided as copper acetate and copper citrate, at four concentrations (10, 25, 50, and 100 mu M) containing 0.64, 1.59, 3.18, 6.35 mu g ml(-1) Cu2+, and 1.91, 4.77, 9.53, 19.06 mu g ml(-1) Cu2+, respectively. SOD and POD showed similar patterns, with relatively small fluctuations compared to control. Considerable rise of activity was registered only in 25 mu M copper citrate (increase rate of +152.90% for SOD, and +70.51% for POD). CAT activity was lower than control in all variants, the smallest level being in 25 mu M copper citrate-treated variant (decrease rate of -65.41%). Generally, copper had negative repercussions on soluble protein level. Higher SOD and POD activities and CAT decline in all copper-treated variants indicate that SOD and POD play a more important role than CAT in preventing copper-induced oxidative stress, in the studied wheat cultivar.

Abstract

Large morpho-anatomical and biochemical variability was evidenced in all Lonicera caerulea L. provenances. The knowledge of chromosome constitution is of basic importance in this context of elevated phenotype heterogeneity and represents a necessary step in breeding programs. The Romanian selections are tetraploid (2n = 4x = 36). Chromosomes are small-sized (<3.5 mu m), and lengths of haploid complements are close (28.97-29.05 mu m). Only the Lc-SL15 complement is larger (33.89 pm), possibly due to lower chromatin condensation. Karyotypes contain metacentric (85%-100%) and submetacentric chromosomes. According to asymmetry indexes (AsI% = 55.01-59.89, TF% = 40.13-44.98, A(1) = 0.17-0.29, A(2) = 0.23-0.30) and to Stebbins' classification, the karyotypes fall into 1B and 2B categories, considered relatively symmetric and primitive in this system. In the idiogram constructed on average values of cytogenetic parameters of all analyzed selections, the mean chromosome length is <(x)over bar> +/- SE = 1.66 +/- 0.07 mu m, ranging from 2.73 +/- 0.20 to 1.10 +/- 0.02 mu m. The mean relative length is between 9.05 +/- 0.30 and 3.70 +/- 0.12. One submetacentric chromosome pair is present. As a result, the general karyotype formula is K(2n) = 36 = 34 m + 2 sm.

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Copper is one the most abundant heavy metals in agricultural soils and its excess in soil comes from the largely use of this heavy metal in industry and agriculture (as fungicide). Mitotic index, rate and categories of ana-telophase chromosome aberrations, as well as the frequency and types of metaphase disturbances were scored in root tip meristems of Triticum aestivum L. cv. Beti after seed exposure to copper, provided as copper acetate and copper citrate, at four concentrations (10, 25, 50, and 100 mu M) containing 0.64, 1.59, 3.18, 6.35 mu g Cu2+, and 1.91, 4.77, 9.53, 19.06 mu g ml(-1) Cu2+, respectively. Except the mitostimulant effect of 25 mu M concentration, all the other concentrations of copper acetate and copper citrate showed mitodepressive action. The copper genotoxicity is expressed in the increased level (1.5 - 5-fold higher than in control) of the rate of chromosome aberrations in mitotic ana-telophases of copper-treated variants. Chromosome bridges, laggards and complex aberrations are the most numerous, although multipolarity, fragments and micronuclei are present, but with lower frequency and not in all copper-treated variants. Concerning the rate of metaphase disturbances, copper acetate augmented 2 3 times the rate of abnormalities in all variants, whereas only variant treated with 25 mu M copper citrate exceeded the control in a substantial manner. Metaphases with chromosomes expulsed from equatorial plate are numerically preponderant, followed by C-metaphases. These observations constitute a signal about the risks of the widespread and increasing presence of some heavy metals into environment. The results reported here could be considered in a future evaluation of copper effects on other organisms, even on human health, due to large use of copper compounds, inclusively as fungicides.

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The sclerotia of the pyrenomicete Claviceps purpurea (Fr.) Tul. (Hypocreaceae) contains numerous active compounds of which the most known are ergoline alkaloids. In this paper, the mycelian extracts and the corresponding concentrated supernatants, separated from cultures of nine Claviceps purpurea strains, have been biochemically investigated by determination of alkaloids, glucans and proteins. The cytostatic potential of extracts and concentrated supernatants has been also tested on HeLa tumor cells cultures in the presence of bioactive samples. Generally, the cytostatic potential of these bioactive agents was inversely correlated with the intra - or extracellular ergoline alkaloids content, which seemed not to be the exclusive biochemical substratum for this in vitro pharmacodynamic effect.

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The somatic chromosomes and karyotype traits of three Romanian genotypes (coded as CB-1, S-16, L-4) of Hippophae rhamnoides L. have been studied. Diploid chromosome number is 2n = 24. The karyomorphological data show that the complements have small chromosomes (1.33+/-0.014-2.80+/-0.01 mu m, for CB-1 genotype; 1.13+/-0.029-2.90+/-0.029 mu m, for S-16; 1.15+/-0.099-2.88+/-0.024 mu m, for L-4 genotype) and the length of haploid complements is 23.97 mu m, for CB-1, 22.20 mu m, for S-16, 21.73 mu m, for L-4. In CB-1 and S-16 male genotypes, the putative Y-chromosome has larger sizes than X-chromosome and it is of metacentric type (CI = 45.77, r = 1.18, for CB-1; CI = 46.22, r = 1.15, for S-16). The L-4 female karyotype is constituted by twelve pairs of morphologically similar chromosomes. The karyotype formulae are 2n = 24 = 13m + 11sm (putative male: 22 + XY), for CB-1 genotype, 2n = 24 = 18m + 6sm (putative male: 22 + XY), for S-16 genotype, respectively 2n = 24 = 22m + 2sm (L-4 genotype, considered as female: 22 + XX). The small size of chromosomes (<3 mu m), the presence of only two morphometric chromosome types and preponderance of metacentrics confer a relatively high degree of symmetry to the studied karyotypes. Our results show a low intraspecific karyotype variation.

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There is great evidence regarding the beneficial influence of rose hips on human and animal health. In this respect, fruits of native populations of Rosa spp. collected from the northeastern part of Romania (400-1060 in altitude) were assayed for nutritional and some morphological properties, in order to select the best genotypes. The results show a great quantitative variability of analyzed genotypes, regarding dimensions and biochemical indicators as follow: ascorbic acid: 866.91 mg/100 g fw (R. rubiginosa), 615.98 mg/100 g fw (R. carrier); carotenes: 34.95 mg/100 g fw (R. subcanina), 24.64 mg/100 g fw (R. nitidula); protein content: 0.47 g/100 g fw (R. micrantba), 0.25 g/100 g fw (R, caesia); total sugars: 17.63 g/100 g fw (R. andegavensis), 11.55 g/100g fw (R. caryophyllacea). The fruits length, width and dry weight were: 25 mm (R. canina), 12 mm (R. micrantha), 16.5 mm (R. corymbifera), 8 mm (R. micrantha) and 25% dry weight (R. rubiginosa), 3896 dry weight (R. nitidula), respectively. The present study demonstrates the existence of a rich rose germplasm source in north-east area, with good biotechnological fruit quality, which recommends them as functional foods according to their chemical contents (R. rubiginosa, R. vosagiaca, R. subcanina, R. canister and R. caryophyllacea) and also as sources for natural colorants in food technology.

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The wide utilization of plant growth regulators requires the knowledge of possible unwanted effects induced by uncontrolled administration of these chemicals, inclusively at genetic level. The cytogenetic effects induced by two plant growth regulators, an auxin (2,4-D) and a cytokinin (kinetin), in root meristems of plants belonging to two species of economic importance - Raphanus sativus L. and Phaseolus vulgaris L. - were studied. Mitotic index, rate and categories of ana-telophase chromosome aberrations, as well as the frequency and types of metaphase disturbances were comparatively analyzed after seed exposure to two concentrations (1 mg/L and 10 mg/L) for each plant hormone. 2,4-D had a slight cytotoxic effect in radish, but did not show genotoxic potential, whereas kinetin stimulated cell division. In common bean, the small concentrations of 2,4-D and kinetin enhanced mitosis, but the maximum tested concentrations had inhibitive effect on cell division. Generally, the rate of ana-telophase chromosome aberrations induced by 2,4-D and kinetin, at the tested concentrations and at the respective exposure, was not significant. Auxin caused increase of total abnormal metaphases (metaphases with expulsed chromosomes and colchicine-like metaphases) in both species, indifferently of concentration, whereas all the variants exposed to kinetin action showed values lower than control, in both species. Therefore, 2,4-D have aneugenic potential by acting as spindle poison and by disturbing the correct separation of the chromosomes to cell poles. The most complex pattern of ana-telophase and metaphase modifications was present in variant of common bean exposed to the action of 10 mg/L 2,4-D.

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