Volume 70, Issue 8

In our work, microbial diversity of Krubera-Voronja cave was evaluated in the view of the frequency of human visits in different locations as well as the sampling depth. Sampling in this cave was performed at depths of 220 m to 1640 m. Cultivation-independent method, namely barcoded pyrosequencing of 16S rRNA gene, was used for this analysis. Our results demonstrated high bacterial diversity at the phylum and genus levels. We have shown that the bacterial diversity at the phylum level depends on both the sampling depth and the frequency of human visits in Krubera-Voronja cave. Frequently visited locations were more diverse at the phylum level than the rarely visited branches. The total number of bacterial genera both per phylum and per sample correlated with the frequency of human visits but not with the sampling depth. Some genera, found in Krubera-Voronja cave, seem to be absent or very rare in other caves. The present study represents the first report on the microbial diversity in Krubera-Voronja cave

Two food-grade antimicrobial substances, thymol and nisin were tested for their antimicrobial activity against 4 species of Shigella including S. boydii, S. dysenteriae, S. flexneri and S. sonnei inoculated in sugarcane juice at 4◦C. When used separately, only thymol, but not nisin, exhibited a dose-dependent and species-dependent inhibitory effect on the bacteria. When thymol and nisin were used together, the concentrations of thymol required for complete inhibition were decreased in all cases of the bacteria as the concentrations of nisin were raised, indicating the synergistic antimicrobial activity between both compounds. The effects of bacterial cell number and temperature on the antimicrobial activity of thymol and nisin against S. sonnei in sugarcane juice were investigated. The doses of both compounds required for complete inhibition were directly proportional to the number of bacterial cells in sugarcane juice. Furthermore, they inhibited the bacterium at 30◦C more efficiently than at 4ºC. The sensory evaluation showed that thymol- and nisin-treated sugarcane juice was acceptable to consumers. This study suggests that thymol and nisin can be used as food preservatives with the consideration of their dependence on bacteria species, number of contaminated bacterial cells and temperature

The aimof the study was to assess the effects of low-temperature hardening (2◦C) on the biochemical compounds and processes that can increase resistance of winter rape to inoculation with Leptosphaeria maculans spores. The study involved an evaluation of the entire pool of phenolic compounds, L-phenylalanine ammonia lyase (PAL) activity, excitation intensity for blue and green fluorescence, catalase (CAT) activity, respiration intensity and heat emission from leaf tissues. All the measurements were performed 24 and 72 hours after the inoculation. Low-temperature hardening, which preceded the inoculation of rape seedlings with spores of L. maculans, caused a significant increase in CAT activity and the level of phenolic compounds. The observed changes in PAL activity reflected the changes in phenolics content. The hardened plants showed a significantly higher intensity of blue fluorescence excitation at 24 and 72 hours after the inoculation, as compared to the non-hardened seedlings. Increased content of phenolic compounds and PAL and catalase activity triggered by the temperature of 2◦C and maintained for 24 hours after the inoculation, may confirm the stimulating effect of the hardening temperature. Intensified emission of blue fluorescence indicating saturation of a cell wall with phenolic compounds makes the cell wall structure less stretchy, more tight and leakproof, and thereby hinders fungal growth through plant tissue.

Mitochondrial morphology and evolution have been observed during seed germination and early seedling development in Arabidopsis thaliana line 43a9 (ecotype Columbia) expressing green fluorescent protein in these organelles. Fluorescence, confocal and electronic microscopy images reveal that mitochondrial development goes through different stages, and that the organelle structure varies with cell types during these processes. Mitochondria develop from larger, isodiametric structures pre-existent in the dry seed called promitochondria. After germination, variations in mitochondrial morphology occur synchronously with cell differentiation and cell division in the course of early root development. Some promitochondria develop into intermediate structures resembling the syncytial organelles. These structures have been described in certain plants under hypoxia as intermediates for the formation of mature mitochondria. On the other hand, other promitochondria temporarily remain in the cells of the root apex

Indigo is the most important blue dye for textile dyeing and is biosynthesized in Polygonum tinctorium. Some biochemical studies related to biosynthesis are available. However, genomic and transcriptome studies have not received sufficient attention. Here, we report de novo assembly of transcriptome datasets and its comprehensive analysis. A total of 60,395 unigenes were annotated using BLAST search against the different databases. At least 23,721 unigenes mapped onto different pathways using KEGG database. We found that 3,323 genes are involved in biosynthesis of secondary metabolites, 117 phenylalanine, tyrosine and tryptophan biosynthesis and 147 tryptophan metabolisms. Apart from this, indigo biosynthesis pathway genes viz., dioxygenase, monooxygenase, and glucosyltransferase have also been identified. Fourteen genes encoding cytochrome P450 monooxygenase, 26 glucoside dioxygenase, 9 UDP-glucose D-glucosyltransferase and 52 were β-D-glucosidase. These findings provide a foundation for further analysis of this pathway with potential to enhance the synthesis of indican in P. tinctorium

Riparian ecosystems host an high level of biodiversity but anthropic activities have deeply altered their naturalness and functionality. The present study was carried out in a protected area along a recently regulated tract of the upper River Tiber (Tuscany, central Italy). The study’s aim is to explore different components of plant diversity (species richness, species composition, β-diversity) in the riparian habitats to determine the most relevant conservation issues. Twelve transects were allocated along the riparian zone and a stratified random sampling was performed on the habitat detected along the transects with 184 plots of 1 x 1 m. Species richness was analysed by the use of set of species-richness estimators. The different patterns of plant diversity were analysed by NMDS, Indicator Species Analysis and SDR with symplex analysis. Five habitat types were detected in the study area. Three well-represented habitats (riparian woodlands, shrublands and dry bars) showed a higher number of species; flooded banks and marshlands were much less common and were also less species rich. Species composition and indicator species analysis showed however that marshlands were the richest in taxa having high conservation value and in indicator species. The habitats differed greatly in species composition; β-diversity analysis showed a low number of shared species between the habitats. High values of species replacement and low similarity values were recorded between the plots within the habitats. The study shows some differences between quantitative and qualitative patterns of biodiversity in the study area. The results suggest that to maintain habitat species diversity, the best conservation strategy for the study area is to protect as large an area as possible

Stipa lagascae R. & Sch. (perennial bunchgrass) is one of the most promising steppic species for arid and desert lands of Tunisia. The present study was designed to study the effect of drought on root and leaf anatomy, water relationship, and the growth of three- month-old S. lagascae plants, submitted to water deficit (5, 10, 15, 20, 30 days of withheld irrigation) and grown in pots in greenhouse conditions. The results show that water deficit treatments reduced the biomass accumulation (MS) and leaf water potential (Ψw) of plants. However, leaf relative water content (RWC) decreased significantly only at severe drought. The root’s anatomical features showed reduced root cross-sectional diameter under water deficit. Conversely, epidermis was unaffected by water stress. Moderate and/or severe water deficit (20-30 days) reduced significantly the cortex thickness, cortical cell size, stele diameter, xylem vessel diameter and the stele/root crosssectional ratio, while the number of cortical cells increased for severe water deficit. The cuticles and mesophyll of S. lagascae was thickened by moderate to severe drought and the entire lamina thickness was increased significantly by 5.8% only after 30 days of water deficit while epidermis was unaffected by water deficit. However, severe water deficit (30 days) decreased the width and the length of the bundle sheath. At the same time, the mesophyll cells size and both the xylem and phloem vessels diameter diminished by 12, 16.8 and 17.5%, respectively. Leaf rolling occurs as a response to water deficit and its level increases as the drought period is progressing in plants while reduced bulliform cells size occurred only at severe water deficit. Our findings suggest a complex network of root and leaf anatomical adaptations such as a reduced vessel size with lesser cortical and mesophyll parenchyma formation and increased leaf rolling. These proprieties are required for the maintenance of water potential and energy storage under water stress which can improve the resistance of S. lagascae to survive in extremely arid areas

Callus cultures of Arnebia euchroma (Royle) Johnst can produce considerable amounts of shikonin derivatives, which are shown to possess a wide spectrum of pharmaceutical properties, such as antimicrobial, anticancer, antioxidative, immune regulative and anti-inflammatory. To explore the possible regulation mechanism of shikonin derivatives biosynthesis, the effects of ammonium ion (NH + 4 ) on cell growth and formation of shikonin derivatives in callus cultures of A. euchroma were investigated in this paper. The main results include: When A. euchroma callus were cultured in medium with different concentrations of NH + 4 (0 mM, 7.02 mM, 14.04 mM), the fresh weight of the cell cultured in 7.02 mM NH + 4 was higher than those in 0 mM and 14.04 mM NH + 4 , indicating that NH+4 is necessary for the cell growth of A. euchroma callus cultures, while excessive NH + 4 concentration (14.04 mM) could rather inhibit the cell growth of A. euchroma. The quantitative analysis of the seven main naphthoquinone compounds showed that the contents of most of the shikonin derivatives (including the bioactive components acetylshikonin and β,β-dimethyl-acrylshikonin) of the Red Strain cultured in 0 mM NH + 4 condition were higher than those in other NH + 4 conditions (7.02 mM, 14.04 mM) at most of the sample points, suggesting that NH + 4 essentially plays an unfavorable role in the biosynthesis of shikonin derivatives. The analysis of quantitative real-time RT- PCR showed that a higher NH + 4 concentration inhibited the expression of key enzyme genes (such as AePGT) of shikonin derivatives biosynthesis, while -NH + 4 condition was conducive to the expression of such genes. In sum, our results show that NH + 4 is necessary for the cell growth of A. euchroma, while unnecessary, or even adversary to the biosynthesis of shikonin derivatives.

The main objective of the present study was to clone, heterologously express and characterize a novel cell wall invertase (FCWI) from a Cu tolerant population of Elsholtzia haichowensis. The full-length FCWI cDNA contained an open reading frame (ORF) of 1671 bp which encoded a 556-amino-acid protein. The theoretical molecular mass and pI of the deduced protein were 62.5 kDa and 9.29, respectively. Phylogenetic analysis showed that FCWI had a closer evolutionary relationship to cell wall invertase of dicot. FCWI was expressed in methylotrophic yeast Pichia pastoris and purified to near homogeneity. Recombinant FCWI enzyme had pH optima of 4.0 and temperature optima of 50◦C. Activity analyses in the presence of various metal cations indicated that FCWI was completely inhibited by Hg 2+ (0%), while retained 77.4% activity when exposure to Cu 2+ . The K m and V max values of FCWI for hydrolyzing sucrose were 0.282 mM and 1.576 μkat/mg, respectively. This is the first report that the heterologous expression and characterization of a cell wall invertase from a Cu tolerant population of E. haichowensis. These results helped to understanding the characteristics of FCWI and its physiological role in the resistance mechanisms of Cu tolerant plants

The molecular mechanisms underlying cold-resistance in Prunus campanulata Maxim. (P. campanulata) are not fully understood. This study aimed to establish a full-length library and analyze expressed sequence tags (ESTs) to provide tools to investigate the mechanisms of P. campanulata growth at low temperatures. Based on the switching mechanism at 5’end of RNA transcript technology, a full-1ength cDNA library was generated from young leaves of P. campanulata after 72 h treatment at 1◦C, and a preliminary EST analysis was carried out. Quantitative reverse transcription polymerase chain reaction was used to assess the expression of selected cold-related genes. The cDNA library titer was 1.2 × 106 cfu/mL−1, with a recombinant rate of 96%. The average size of inserted cDNA fragments was 1.3 Kb. EST data revealed the existence of 834 clones representing a total of 667 unigenes, including 574 singletons and 93 contigs. Blast analysis identified 475 unigenes with known and putative functions. Based on similarity search and GO annotation, 84 unigenes were associated with “response to stimuli”, suggesting that cold stress induced significant alterations in gene expression in P. campanulata cultivated at 1◦C for 72 h. Interestingly, DRP, MYB, HSP, GPX and GA20-ox gene expression was significantly up-regulated in plants cultivated at low temperature, while transcript levels of TIL, CDPK were decreased. P. campanulata cultivating at low temperature express genes associated with “response to stimuli”, and in particular DRP, MYB, HSP, GPX and GA20-ox gene are up-regulated while TIL, CDPK are downregulated in response to low temperature-stress

Architectonics of the leech body constitute a source of promorphological characters which have been used for modeling. A geometric model of the leech body form was applied in a phylogenetic analysis for the estimation of the utility of morphometric data in the reconstruction of the history of Hirudinida. A cladistic analysis included species representing all main ecological groups of leeches. In the analysis the morphometric data were used as non-discretized mean values and as mean values coded with the gap-weighting method. Obtained phylogenetic trees were compared with cladograms generated based on combined data sets including discrete qualitative morphological characters as well as known molecular phylogenies of leeches. The study demonstrates that the morphometric characters provide additional evidence for the confirmation of monophyletic groups within Hirudinida. Thus, the morphometric data resulting from the geometric model of the leech body form appeared to be applicable for the reconstruction of leech phylogeny

The aims of this study were to determine the substrate choice by the alien snail Ferrissia fragilis (Tryon, 1863) (Phragmites australis and Typha latifolia remains, Nuphar lutea floating leaves and leaf deposit) as well as the environmental factors that influence the occurrence of this species. From 9 (T. latifolia and P. australis) to 11 (leaf deposit) snail species occurred on each type of substrate. F. fragilis belonged to the eudominants on most of the substrate types. This species reached its highest average density on floating N. lutea leaves (271 ind. m−2) and the lowest was on P. australis (60 ind. m−2). It occurred in different periods during the snail collection: in the initial study period on the floating N. lutea leaves (May) and on P. australis in the final period of the study (September). In July, we found a few specimens in the postseptal form on T. latifolia remains. The results of RDA analysis confirm the association between F. fragilis and T. latifolia as well as with floating N. lutea leaves. F. fragilis occurs at sites that have a higher content of calcium and iron in the water. Alien species more increasingly appear in disturbed anthropogenic water-bodies. An important vector in the dispersion of F. fragilis was probably the water birds or the economic activities that are related with the use of this forest pond. Studies on the occurrence of alien species contribute important data on the activities that are taken to preserve the biodiversity of freshwater environments

The composition of the land snail fauna of central Europe is responsive to environmental conditions and undergoes changes throughout the Holocene. That holds also for the fauna of central-European Helicellinae, a group of dry-adapted western Palearctic snails with five species living in the Czech Republic. While one of them is currently spreading in the country, two, Candidula unifasciata and Helicopsis striata, are critically endangered, both with only a single surviving population. Although sharing a similar fate lately, these two species represent completely different histories. While C. unifasciata is a rather recent immigrant in the area, H. striata is a relict species of the Pleistocene loess steppe, which is declining during the Holocene. However, the origin of the only known extant Czech population of H. striata is deemed to be dubious due to the untypical habitat inhabited, and within C. unifasciata two species or subspecies used to be distinguished, suggesting a dual origin of its populations. We present here sequences of a fragment of the 16S rRNA gene from two extinct and the last surviving populations of H. striata, and from two extinct populations of C. unifasciata from Bohemia, supplemented by Slovak samples of both. The obtained haplotypes of Bohemian C. unifasciata are different from the one found in western Slovakia, suggesting a different origin of the Western Carpathian populations. The sampled extinct Bohemian populations of H. striata shared an identical haplotype with the last living population, supporting native origin and conservation value of the latter. The causes of the disappearance of the two species are discussed

Monteustium marezensis gen. n., sp. n. is described and Italustium eframi Haitlinger is new to the fauna of Montenegro. New metric data for I. eframi are given

Lithobius (Ezembius) laevidentata sp. n. (Lithobiomorpha: Lithobiidae), discovered from Barkol Kazak Autonomous County, Hami City, Xinjiang Uygur Autonomous Region, Northwest region of China, is described. Morphologically it resembles L. (E.) bilineatus Pei, Ma, Zhu & Gai, 2014. from Guangxi Zhuang Autonomous Region, China, but can be readily distinguished from the latter by 8–10 ocelli arranged in 3 irregular rows, Tömösváry’s organ subequal in size to the adjoining ocelli, female gonopods with 3–4 spurs, and the terminal claw of the third article of female gonopods simple. A key to Chinese Lithobius (Ezembius) species is presented.

The aim of this project was to characterize the aphid groups closely, relating to xerothermic plant assemblages (Festuco-Brometea class) and identifing the aphid species diagnostic for this type of community (aphidocoenosis). The paper represents the first attempt to classify the aphid communities distributed in one type of particular habitat at a country-level scale. The research involved quantitative and qualitative sampling, conducted at 23 xerothermic study sites in 7 regions (belonging to 3 geographical provinces of Central Europe), for three years. Among the 187 aphid species collected, 91 were determined to be closely associated with xerothermic habitats. Of these, 32 were considered to be diagnostic in recognizing xerothermic aphid communities. The xerothermic habitats in question proved to be diverse, with a high value of Shannon’s index (Pielou’s eveness index) (85.95%). The diagnostic species formed a characteristic species community, varying between the regions of the studied areas. This was confirmed by a statistical analysis of similarities based on Ward’s method. Frequency analyses indicate that many aphids related to xerothermic habitats are scarce locally and rare on a regional spatial scale. They exhibit a narrow geographical range and a narrow ecological amplitude and thus can be described as unique on a global scale

The winter raptor community was studied in river valleys of North Eastern Poland during the winters of 2000- 2010. Via transect counts method we showed the potential drivers of raptor abundance, and co-occurrence patterns. We analysed the influence of weather and habitat factors along on the single species abundance and diversity of the raptor assemblage. We found that higher temperatures corresponded with higher occurrence of the common buzzard Buteo buteo, rough-legged buzzard Buteo lagopus, Eurasian sparrowhawk Accipiter nisus, hen harrier Circus cyaneus and lower of the common kestrel Falco tinnunculus. When considering the effect of each habitat variable on the whole raptor assemblage, the relative proportion of urban areas to pasture had the most distinct effect on species abundance and diversity. Most of species were found to be less abundant in areas with a high proportion of urban areas, while some, like the Eurasian sparrowhawk and the northern goshawk Accipiter gentilis, occurred more often on such areas. We observed that the difference in the number of these species could be explained mainly by the different habitat requirements due to individual food preferences and flexibility of migration strategies