Volatile and molecular analysis of Juniperus brevifolia (Seub.) Antoine,an Azorean endemic species

Chemical and molecular analyses were performed for the characterization of Juniperus brevifolia (Seub.) Antoine (Cupressaceae), an Azorean endemic species. Forty-two individual twig samples were collected at seven Azorean islands (S. Miguel, Terceira, S. Jorge, Pico, Faial, Flores and Corvo). Volatile compounds were isolated by distillation–extraction and analyzed by GC and GC-MS. The volatile oils consisted mainly of monoterpene hydrocarbons (84–96%), limonene (33–87%), α-pinene (1–48%) and β-myrcene (1–5%) being the main components, and cluster analysis showed a high correlation among all samples (S_corr = 0.84). DNA fingerprinting was performed using thirty-seven RAPD and eleven ISSR primers, generating 881 polymorphic bands. Cluster analysis showed a moderate correlation among accessions (S_Dice = 0.43), which grouped according to their geographical origin. Chemical and molecular data did not show superimposable clustering profiles. Although the molecular approaches tested were useful in assessing genetic diversity, no straight correlation could be drawn between chemical and molecular data sets.     

Genetic diversity and chemical polymorphism of Thymus caespititius from Pico,São Jorge and Terceira islands (Azores)

Thirty-one individuals from Thymus caespititius were collected on the islands of Pico, São Jorge and Terceira (Azores) and the volatiles were isolated by distillation–extraction and analyzed by GC and GC–MS. The same plants were analyzed by RAPD using 17 arbitrary primers that generated 187 scorable polymorphic bands. The volatile composition of the individuals was in accordance with previous results obtained from the populations’ analysis, confirming that the chemical polymorphism was in some cases more evident among different plants from the same island than among those collected on different islands. RAPDs analyses using the same individuals did not show identical clustering as with the volatile oil profiles and no straight correlation among collection site, chemical analysis and molecular assessments could be found, suggesting that other molecular tools should be explored in order to fully understand the influence of both environmental and genetic factors on volatiles composition.     

Chemotaxonomy of Hypericum genus from Portugal: Geographical distribution and essential oils composition of Hypericum perfoliatum, Hypericum humifusum, Hypericum linarifolium and Hypericum pulchrum

The geographical distribution and analysis of the essential oils of species from three sections of Hypericum L. (Guttiferae/Clusiaceae/Hypericaceae) from Portugal are presented. Hypericum perfoliatum (section Drosocarpium) grows wild in the centre and south of Portugal; Hypericum humifusum and Hypericum linarifolium are both from section Oligostema, the former occurring throughout the country, while the second is distributed mainly in the north and centre; Hypericum pulchrum (section Taeniocarpium) is confined to the littoral north of Portugal. The essential oils were obtained by distillation–extraction, hydrodistillation and distillation in a modified Marcusson apparatus from the dried aerial parts of the different populations and were analysed by GC and GC–MS. Monoterpene hydrocarbons constituted the main fraction in all oils (43–69%, 53–85%, 28–45% and 48–65% for H. perfoliatum, H. humifusum, H. linarifolium and H. pulchrum, respectively). Sesquiterpene hydrocarbons (2–13%, 6–18%, 21–27% and 16–18%, respectively) and a third fraction of non-terpenic compounds (20–29%, 3–16%, 2–14% and 5–11%, respectively) from the four species attained relatively high amounts in all oils. Within each species, no major differences were detected in the essential oil composition, despite the fact that different locations, phenological phases and extraction methodologies were used. Notwithstanding the dominance of α-pinene in all four species' oils, cluster and principal components analysis on the identified components showed that the range of α-pinene, β-pinene and n-nonane supported a separation of the four species. The essential oil composition of the four species showed some qualitative resemblances, which correlate well with the taxonomical classification based on morphological characters.     

Terpenoids from the seed of Thujopsis dolabrata var. Dolabrata

Fourteen mono-, 13 sesqui-, 16 diterpenes, dodecanal, sitosterol and desoxypodophyllotoxin were identified in the seed of T. dolabrata var. dolabrata. Sabinene and α-pinene were found to be the main components of the volatile oil. From the diterpenoid fraction, two new abietane-type compounds (ar-abietatrien-12,16-oxide and 16-hydroxyferruginol) were isolated and their structures were elucidated. Significant differences were observed in the seed diterpenoids when these results were compared with those obtained earlier with T. dolabrata var. hondae.     

Description of Brenneria roseae sp. nov. and two subspecies, Brenneria roseae subspecies roseae ssp. nov and Brenneria roseae subspecies americana ssp. nov. isolated from symptomatic oak

Gram-negative, facultatively anaerobic bacteria were isolated from symptomatic oak tissue in the UK and USA. Partial gyrB sequencing placed ten strains in the genus Brenneria, with B. goodwinii as the closest phylogenetic relative. The strains were investigated further using a polyphasic approach including MLSA (based on partial gyrB, rpoB, infB and atpD gene sequences), 16S rRNA gene sequencing, DNA–DNA relatedness studies and both phenotypic and chemotaxonomic assays. The MLSA and 16S rRNA gene analyses separated the strains into two groups based on origin, suggesting that they belong to Brenneria as two novel species. However, the DNA–DNA relatedness values revealed a closer relationship between the groups and indicated that they should belong to the same species. As the two groups of strains from the UK and USA can be differentiated from each other phenotypically and by ERIC PCR fingerprints, it is proposed to classify them as novel subspecies of a novel Brenneria species. The name Brenneria roseae sp. nov. (FRB 222^T = LMG 27714^T = NCPPB 4581^T) is proposed, with Brenneria roseae subsp. roseae ssp. nov. (FRB 222^T = LMG 27714^T = NCPPB 4581^T) for the strains from the UK and Brenneria roseae subsp. americana ssp. nov. (FRB 223^T = LMG 27715^T = NCPPB 4582^T) for the strains from the USA.     

Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene

RIG-I (retinoic acid-inducible gene I) is an essential cytosolic pathogen recognition receptor that binds to a variety of viral RNA or DNA to induce type I interferons. In the present study, insert–deletion polymorphisms in promoter and introns of CiRIG-I (Ctenopharyngodon idella RIG-I) were explored, their associations with resistance/susceptibility to grass carp reovirus (GCRV) were analyzed. To this end, genomic sequence of CiRIG-I gene was obtained, and twenty pairs of primers were prepared for the detection of insert–deletion polymorphisms. Five insert–deletion mutations were found, a 2-bp mutation and an 8-bp mutation existed in the promoter and other three sizes in 74 bp, 146 bp and 53 bp were sited in the intron 8. After a challenge experiment, only the genotype and allele of − 740 insert–deletion mutation in the promoter and allele of 6804 insert–deletion mutation were significantly associated with resistance/susceptibility to GCRV among the five mutations (P < 0.05). To further identify this correlation, another independent challenge test was carried out. The result revealed that the cumulative mortality in ins/ins genotype individuals (43.75%) at − 740 insert–deletion mutation was significantly lower than that in ins/del (72.09%) and del/del (74.19%) genotypes (P < 0.05). Linkage disequilibrium and haplotype analysis showed 6610 insert–deletion mutation and 6804 insert–deletion mutation were linkage disequilibrium. The haplotype ins–ins (6610ins–6804ins) was significantly susceptible to GCRV, and ins–del (6610ins–6804del) was significantly resistant to GCRV (P < 0.05). Those could be potential gene markers for the future molecular selection of strains that are resistant to GCRV.     

Chemical composition,intraspecies variation and seasonal variation in essential oils of Calendula arvensis L.

The essential oil composition of Calendula arvensis was established for the first time using GC and GC/MS. Eighty-five essential oil components were identified, which accounted for 90.3 g/100 g of essential oil. The oil contained a high concentration of sesquiterpenes, of which δ-cadinene and α-cadinol were the main components. The chemical composition of 25 Corsican C. arvensis oils was analyzed to determine intraspecies variation in essential oil composition. A matrix linking essential oil composition to sample location was composed to identify relationships between concentrations of volatile samples and the geographical origins of samples. Two main groups of compounds were identified according to the amount of sesquiterpenic compounds (hydrocarbons and alcohols) and soil characteristics. Seasonal variation (winter vs. spring) in the concentrations of two major compounds during the flowering period was observed.     

Pseudoscardovia suis gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of wild pigs (Sus scrofa)

Seventeen fructose-6-phosphate phosphoketolase-positive bacterial strains were isolated from the digestive tract of wild pigs (Sus scrofa). Most of them were identified as Bifidobacterium boum according to sequences of 16S rRNA gene. Two strains isolated from the small intestine content had unusual morphology of cells in comparison with bifidobacteria. Cells growing in liquid anaerobic media were regular shaped rods arranged mostly in pairs. These isolates showed relatively low 16S rRNA gene sequence similarities (maximum identity of 94%) to members of the family Bifidobacteriaceae. Nevertheless, phylogenetic analyses of 16S rRNA, hsp60 and xfp gene sequences revealed that these strains are more related to recently described Neoscardovia, Aeriscardovia and other scardovial genera, than to Bifidobacterium species. Partial gene sequences of other phylogenetic markers showed low (65.8–89.5%) similarities to genome sequences of bifidobacteria and Gardnerella vaginalis. The major fatty acids detected in cells of the representative strain DPTE4^T were C_16:0, C_18:1, C_14:0. The peptidoglycan type of the DPTE4^T strain was A3β l-Orn(l-Lys)-l-Ser(l-Ala)-l-Ala₂. Polar lipid analysis revealed two phosphoglycolipids and phospholipids, a glycolipid and diphosphatidylglycerol. The results of phylogenetic, genotypic and phenotypic analyses support the proposal of a novel taxa, Pseudoscardovia suis gen. nov., sp. nov. (type strain = DPTE4^T = DSM 24744^T = CCM 7942^T).     

Seed bank,biomass, and productivity of Halophila decipiens, a deep water seagrass on the west Florida continental shelf

One of the largest contiguous seagrass ecosystems in the world is located on the shallow continental shelf adjacent to the west coast of Florida, USA and is comprised of seasonally ephemeral Halophila decipiens meadows. Little is known about the demography of the west Florida shelf H. decipiens, which may produce 4.56 × 10⁸ g C day⁻¹ or more during the peak growing season. We documented seagrass distribution, biomass, and productivity, and density of sediment seed reserves, seedlings, flowers and fruits on the southeastern portion of the west Florida shelf by sampling along a transect at three stations in 10, 15, and 20 m water depth. Biomass, flower, fruit, seedling, and seed bank densities tended to be highest at stations in 10–15 m water depth and lowest at 20 m. Flowers and fruit were most prevalent during summer cruises (June and August 1999, July 2000). Seedling germination occurred during summer, fall (October 1999), and winter (January 2000) sampling events, with the highest seedling densities present during the winter. Seed bank density remained consistent through time. A Category I hurricane with sustained winds of 120 km h⁻¹ passed over the stations, but only limited impact on H. decipiens biomass was observed. The presence of a persistent seed bank provides for recovery after storm disturbance, annual reestablishment of populations, and continual maintenance of the 20,000 km² of deep water seagrass habitat present on the west Florida shelf.     

Haplotype distribution in the GLI3 gene and their associations with growth traits in cattle

The glioma-associated oncogene family zinc finger 3 gene (GLI3) mediates in all vertebrates hedgehog (Hh) signaling that plays an essential role in the induction and patterning of numerous cell types during invertebrate and vertebrate development. In this study, a total of 6 single nucleotide polymorphisms (SNPs: 1–6) were identified by polymerase chain reaction–single stranded conformational polymorphism (PCR–SSCP) and DNA pool sequencing, including all 13 exons and 12 exon–intron boundaries within the bovine GLI3 gene. 16 haplotypes and 13 combined genotypes were revealed and the linkage disequilibrium was assessed in 708 individuals representing three main cattle breeds from China. The statistical analyses indicated that the SNP2, 3 and 4 are associated with the body weight at birth and 6 months in Nanyang cattle population (P < 0.05). No significant association was detected between 11 combined genotypes and body weight at five different ages. Our results provide evidence that polymorphisms in the GLI3 gene are associated with growth traits, and may be used for marker-assisted selection in beef cattle breeding program.     

Scent chemistry and patterns of thermogenesis in male and female cones of the African cycad Encephalartos natalensis (Zamiaceae)

Most, if not all, extant cycads are pollinated by insects which use the cones as larval brood sites. These interactions appear to be mediated by cone volatiles, and, in some species, by patterns of thermogenesis. We investigated the chemical composition of volatile emissions and patterns of thermogenesis in cones of the South African cycad Encephalartos natalensis, using a gas chromatograph–mass spectrometer (GC–MS) and miniature temperature data loggers (ibuttons), respectively. This was done during various developmental stages (before and during receptivity and pollen release) for both female and male cones. A total of 31 compounds were identified in headspace samples; 17 of which were common to both sexes, 12 found only in male cones, and two found only in female cones. The major volatiles in pollen and female cones are (3E)-1,3-octadiene (averaging 54.25% and 15.82% of total emissions), (3E,5Z)-1,3,5-octatriene (averaging 13.37% and 47.66%), and α-pinene (averaging 16.29% and 12.24%). Female cones were not thermogenic before and during receptivity whereas pollen cones were thermogenic during pollen shedding. Thermogenesis of male cones occurred between 1400 h and 1530 h on successive afternoons, reaching an average of c. 10.5 °C above ambient temperature. Volatile emissions and thermogenesis occurred in association with insect activity on the cones suggesting that they both play a role in regulating insect behaviour.     

The relationship between five widely-evaluated variants in CDKN2A/B and CDKAL1 genes and the risk of type 2 diabetes: A meta-analysis

The genes encoding two cyclin-dependent kinases-inhibitor-2A/B (CDKN2A/B) and 5 regulatory subunit-associated protein-like 1 (CDKAL1) have been investigated extensively in associations with type 2 diabetes; the results, however, are often irreproducible. We therefore sought to evaluate these associations by performing a meta-analysis on five widely-evaluated variants from the two genes. There were 38 studies (patients/controls: 51,940/52,234) for rs10811661, 16 studies (20,029/24,419) for rs564398 in CDKN2A/B gene, and 27 studies (28,383/47,635) for rs7756992, 26 studies (28,816/31,713) for rs7754840, 21 studies (29,260/38,400) for rs10946398 in CDKAL1 gene. Overall risk estimates for type 2 diabetes conferred by rs10811661-T, rs564398-A, rs7754840-C, rs7756992-G, and rs10946398-C alleles were 1.17 (95% CI: 1.10–1.23; P < 0.0005; I² = 83.9%), 1.1 (95% CI: 1.0–1.21; P = 0.051; I² = 88.3%), 1.24 (95% CI: 1.18–1.3; P < 0.0005; I² = 74.3%), 1.2 (95% CI: 1.11–1.3; P < 0.0005; I² = 92.0%), and 1.19 (95% CI: 1.1–1.29; P < 0.0005; I² = 90.8%), respectively. There was evident publication bias for rs564398 and rs7754840. Subgroup analyses by ethnicity showed remarkable divergences in risk estimate for rs564398 between Asians (odds ratio [OR] = 1.01; 95% CI: 0.86–1.19; P = 0.868) and Caucasians (OR = 1.19; 95% CI: 1.03–1.35; P = 0.012) (P < 0.05). For all variants examined, the results of studies in retrospective design or with population-based controls were comparative with that of overall studies. In meta-regression analyses, age was found to exert a significant influence on the association between rs10811661 and type 2 diabetes (P = 0.003), as well as between rs7754840 and gender (P = 0.034). Taken together, our findings provide evidence for a significant contribution of CDKN2A/B gene rs10811661 and CDKAL1 gene rs7756992 and rs10946398 to type 2 diabetes.     

In vitro and in vivo screening of essential oils for the control of wet bubble disease of Agaricus bisporus

Proliferation of fungal pathogens, such as Mycogone perniciosa, can severely affect the yields of cultivated mushrooms, including that of the button mushroom, Agaricus bisporus. A reduction in the number of fungicidal products approved for commercial application is currently providing new challenges to the mushroom industry. Forty essential oils, seven pure terpenoids and one phenylpropanoid were screened in vitro to determine the abilities of these substances to inhibit the growth of M. perniciosa. The fungal growth medium of both A. bisporus and M. perniciosa was supplemented with each test substance at a concentration of 50 μL/L. Ten essential oils were further investigated at lower concentrations ranging from 5 to 40 μL/L. The main components of these oils were determined by GC–FID and GC–MS. Lemon verbena (Lippia citriodora), lemongrass (Cymbopogon citratus) and thyme (Thymus vulgaris) oils were found to substantially inhibit the growth of the pathogen, while demonstrating lower toxicity towards A. bisporus than any of the other oils tested. A preliminary in vivo trial using M. perniciosa-inoculated casings revealed that the preventative use of lemon verbena or thyme oils was able to control the development of the disease. A commercial trial using these oils, as well as two of their main components (nerol and thymol), at a concentration of 40 μL/L, revealed that none of these treatments were detrimental to the growth of the A. bisporus and an overall yield similar to that following application of a commercial fungicide (Chronos 450 SC) was obtained. These results suggest that essential oils or mixtures of selected pure components of essential oils may in future find application in button mushroom production, either as a substitute for synthetic fungicides or as an additional protective measure.     

Chemical composition and larvicidal activity of essential oils from Piper species

The larvicidal activity of essential oils of four species of Piper from the Amazon Forest was tested using third-instar larvae of Aedes aegypti. The oils were extracted by steam distillation and analyzed by GC and GC–MS. The main components isolated from each Piper species were as follows: viridiflorol (27.50%), aromadendrene (15.55%) and β-selinene (10.50%) from Piper gaudichaudianum; β-selinene (15.77%) and caryophyllene oxide (16.63%) from Piper humaytanum; dillapiol (54.70%) and myristicin (25.61%) from Piper permucronatum; and asaricin (27.37%) and myristicin (20.26%) from Piper hostmanianum. Amongst all essential oils tested, the most active against larvae of A. aegypti was the oil extracted from P. permucronatum, with a LC₅₀ = 36 μg/ml (LC₉₀ = 47 μg/ml), followed by the essential oil of P. hostmanianum, with a LC₅₀ = 54 μg/ml (LC₉₀ = 72 μg/ml). The oils with higher content of arylpropanoids were more active against larvae of A. aegypti.     

Volatile antimicrobial compounds in the pelage of the Mexican free-tailed bat, Tadarida brasiliensis mexicana

Gas chromatography–mass spectrometry (GC–MS) showed that CH₂Cl₂ extracts of the pelage from the Mexican free-tailed bats, Tadarida brasiliensis mexicana, contained only seven volatile compounds. They include two hydrocarbons, 1-octene and octane; three aldehydes, heptanal, octanal and nonanal; a carboxylic acid, nonanoic acid; and urea. It is likely that nonanal, at an average concentration 0.62 μg/mg hair, and the two homologues, heptanal and octanal found at 0.059 and 0.066 μg/mg hair, respectively, function as antimicrobial agents against mammalian skin bacteria and fungi. These three aldehydes may also act as a chemical protection against bat ectoparasites.     

Bradyrhizobium arachidis sp. nov., isolated from effective nodules of Arachis hypogaea grown in China

Twenty-three bacterial strains isolated from root nodules of Arachis hypogaea and Lablab purpureus grown in five provinces of China were classified as a novel group within the genus Bradyrhizobium by analyses of PCR-based RFLP of the 16S rRNA gene and 16S–23S IGS. To determine their taxonomic position, four representative strains were further characterized. The comparative sequence analyses of 16S rRNA and six housekeeping genes clustered the four strains into a distinctive group closely related to the defined species Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, Bradyrhizobium huanghuaihaiense, Bradyrhizobium japonicum and Bradyrhizobium daqingense. The DNA–DNA relatedness between the reference strain of the novel group, CCBAU 051107^T, and the corresponding type strains of the five mentioned species varied between 46.05% and 13.64%. The nodC and nifH genes of CCBAU 051107^T were phylogenetically divergent from those of the reference strains for the related species. The four representative strains could nodulate with A. hypogaea and L. purpureus. In addition, some phenotypic features differentiated the novel group from the related species. Based on all the results, we propose a new species Bradyrhizobium arachidis sp. nov. and designate CCBAU 051107^T (=CGMCC 1.12100^T = HAMBI 3281^T = LMG 26795^T) as the type strain, which was isolated from a root nodule of A. hypogaea and had a DNA G + C mol% of 60.1 (Tm).