Metabolic fingerprinting of wild type and transgenic tobacco plants by 1H NMR and multivariate analysis technique

The metabolomic analysis of wild type and constitutive salicylic acid producing tobacco plants (CSA tobacco, Nicotiana tabacum 'Samsun' NN) plants overexpressing salicylate biosynthetic genes was carried out by 1H NMR spectrometry and multivariate analysis techniques. The principle component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by PC1 and PC2. The discrimination of non-inoculated, TMV-virus inoculated, and systemic leaves or veins could also be obtained by PCA analysis. Major peaks in 1H NMR spectra contributing to the discrimination were assigned as those of chlorogenic acid, malic acid, and sugars. This method allows an efficient differentiation between wild type and transgenic plants without any pre-purification steps.     

Species delimitation in the European species of Clavulina (Cantharellales, Basidiomycota) inferred from phylogenetic analyses of ITS region and morphological data

The identification of the conventionally accepted species of Clavulina (Cantharellales, Basidiomycota) in Europe (Clavulina amethystina, Clavulina cinerea, Clavulina cristata, and Clavulina rugosa) is often difficult and many specimens are not straightforwardly assignable to any of those four species, which is why some authors have questioned their identity. In order to assess the status of those species, a morphological examination was combined with the molecular analysis of the ITS region. The same six major clades were obtained in the Bayesian and parsimony phylogenetic analyses, and all six clades were well-supported at least by one of the analyses. Morphological characters, such as the overall branching pattern, the presence and intensity of grey colour, the cristation of the apices, and basidiospore size and shape were to various extents correlated with the phylogenetic signal obtained from the ITS region. The congruence between the molecular analyses and morphology, rather than geographical origin, suggests the existence of several species that can be delimited using a combined phylogenetic and morphological species recognition. The analyses revealed that C. cristata and C. rugosa are well-delimited species. In contrast, more than one taxa could be subsumed under the names C. amethystina and C. cinerea, the taxonomical complexity of which is discussed. The ITS region is proved to be adequate to separate phylogenetic species of Clavulina.     

Inter- and intraspecific variation in defensive compounds produced by five neotropical stink bug species (Hemiptera: Pentatomidae)

The differences in composition of defensive secretions between nymphs, adult males and adult females of Chinavia impicticornis (=Acrosternum impicticorne), Chinavia ubica (=Acrosternum ubicum), Euschistus heros, Dichelops melacanthus and Piezodorus guildinii (Hemiptera, Pentatomidae) were analysed within and between species using compositional log-ratio statistics and canonical variates analysis. Differences in composition between nymphs, males and females were found for all species, as well as when all species were pooled. In particular, tetradecanal appears to be a predominantly nymphal compound in D. melacanthus, E. heros and P. guildinii. In the two Chinavia species 4-oxo-(E)-2-hexenal and an unknown compound were more dominant in nymphs. The interspecific analysis revealed a good separation of defensive compounds according to their taxonomic relationship. Thus, the two Chinavia species grouped together, with (E)-2-decenal and (E)-2-hexenyl acetate, contributing to this separation. The other three species also differed from each other, with (E)-2-octenal associated to D. melacanthus, (E)-2-hexenal to P. guildinii and (E,E)-2,4-decadienal and tetradecanal to E. heros. The pooled analysis of stage ignoring species revealed tetradecanal and 4-oxo-(E)-2-decenal (tentative identification) strongly associated to nymphs. Thus, there are predictable differences between stages, and many of the differences are conserved between species. Consideration of these differences could prove to be important in understanding stink bug-natural enemy interactions, and in optimising biocontrol efforts.     

Taxonomic review of the subgenus Uroleucon (Uromelan) (Hemiptera: Aphididae) in the Korean peninsula

Until now, five species of the subgenus Uroleucon (Uromelan) have been recognized in Korea. This is the first report of Uroleucon (Uromelan) adenophorae (Matsumura, 1918) occurring on Adenophora triphylla (Campanulaceae) in Gangwon-do, South Korea. Host plants are reviewed and an identification key to species is presented for six Uroleucon (Uromelan) species from the Korean Peninsula.     

Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae)

The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades - Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66 kDa protein mRNA, clone L5-400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%.     

Relationships between Bambusa species (Poaceae,Bambusoideae) revealed by random amplified polymorphic DNA

RAPD (random amplified polymorphic DNA) molecular markers were used to investigate relationships between a sample of Bambusa species from South Eastern China that have been placed in Bambusa or in several segregate genera, Dendrocalamopsis, Leleba, Lingnania, Neosinocalamus and Sinocalamus by different authors. On the resultant neighbor-joining tree, a thorny core Bambusa cluster was distinguished, as was a Lingnania group, and a cluster of Dendrocalamus species with more capitate inflorescences. However, Leleba was found to be a polyphyletic group in the present study.     

RPB2 sequences reveal a close phylogenetic relationship between tetraploid Hordelymus and diploid Hordeum species in Triticeae (Poaceae)

The origin of Hordelymus genome has been debated for years, and no consensus conclusion was reached. In this study, we sequenced and analyzed the RPB2 (RNA polymerase subunit II) gene from Hordelymus europaeus (L.) Harz, and its potential diploid ancestor species those were suggested in previous studies. The focus of this study was to examine the phylogenetic relationship of Hordelymus genomes with its potential donor Hordeum, Psathyrostachys, and Taeniatherum species. Two distinguishable copies of sequences were obtained from H. europaeus. The obvious difference between the two copies of sequences is a 24 bp indel (insertion/deletion). Phylogenetic analysis showed a strong affinity between Hordeum genome and Hordelymus with 85% bootstrap support. These results suggested that one genome in tetraploid H. europaeus closely related to the genome in Hordeum species. Another genome in H. europaeus is sister to the genomes in Triticeae species examined here, which corresponds well with the recently published EF-G data. No obvious relationship was found between Hordelymus and either Ta genome donor, Taeniatherum caput-medusae or Ns genome donor, Psathyrostachys juncea. Our data does not support the presence of Ta and Ns genome in H. europaeus, and further confirms that H. europaeus is allopolyploid.     

Glucosinolate profiling of Brassica rapa cultivars after infection by Leptosphaeria maculans and Fusarium oxysporum

The glucosinolate contents of two different cultivars of Brassica rapa (Herfstraap and Oleifera) infected with Leptosphaeria maculans and Fusarium oxysporum were determined. Infection triggered the accumulation of aliphatic glucosinolates (gluconapin, progoitrin, glucobrassicanapin and gluconapoleiferin) and indole glucosinolate (4-hydroxy-glucobrassicin) in Herfstraap and of two indole glucosinolates (glucobrassicin and 4-hydroxy-glucobrassicin) in Oleifera. While total and aliphatic glucosinolates decreased significantly in Oleifera, a large increase was observed in Herfstraap after fungal infection. The indole glucosinolate glucobrassicin accumulated in Oleifera at a higher rate than Herfstraap especially after infection with F. oxysporum. Apparently the interaction between fungus and B. rapa is cultivar and fungal species specific.     

基于COI基因全长序列的假眼小绿叶蝉地理种群遗传分化研究

假眼小绿叶蝉Empoasca vitis(Gthe)是我国大陆茶区分布最广、为害最重的茶树害虫,其种群世代重叠严重,数量大。从我国13个主要产茶省份各选出一个重点产茶县(市),采集假眼小绿叶蝉标本,首次扩增得到其线粒体CO I基因全长序列,并以此探讨了假眼小绿叶蝉13个地理种群间的遗传多样性、分子变异、遗传分化程度及基因流水平。在13个地理种群中共得到了176条CO I基因序列,发现了113个变异位点,形成了105个CO I单倍型。总群体单倍型多样性Hd为0.9720,种群内单倍型多样性在0.804—1.000范围内,总群体和各种群的Tajima's D检验结果均不显著,说明茶园假眼小绿叶蝉的进化符合中性模型,种群数量较为稳定。AMOVA分子变异分析结果表明,茶园假眼小绿叶蝉的遗传分化主要来自于种群内部。Mantel检验显示各地理种群的遗传距离与地理距离之间不具有显著的相关性。总群体的遗传分化系数Gst为0.03652,固定系数Fst为0.10876,基因流Nm为4.097,表明地理种群间存在较频繁的基因交流,遗传差异较小。推测,20世纪90年代以来的省际之间频繁的茶树鲜叶贩运、异地茶苗的大批量调拨等因素促进了假眼小绿叶蝉的长距离迁移,加强了不同种群间的基因交流。     

Metabolic classification of South American Ilex species by NMR-based metabolomics

The genus Ilex to which mate (Ilex paraguariensis) belongs, consists of more than 500 species. A wide range of metabolites including saponins and phenylpropanoids has been reported from Ilex species. However, despite the previous works on the Ilex metabolites, the metabolic similarities between species which can be used for chemotaxonomy of the species are not clear yet. In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics was applied to the classification of 11 South American Ilex species, namely, Ilex argentina, Ilex brasiliensis, Ilex brevicuspis, Ilex dumosa var. dumosa, I. dumosa var. guaranina, Ilex integerrima, Ilex microdonta, I. paraguariensis var. paraguariensis, Ilex pseudobuxus, Ilex taubertiana, and Ilex theezans. ¹H NMR combined with principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and hierarchical cluster analysis (HCA) showed a clear separation between species and resulted in four groups based on metabolomic similarities. The signal congestion of ¹H NMR spectra was overcome by the implementation of two-dimensional (2D)-J-resolved and heteronuclear single quantum coherence (HSQC). From the results obtained by 1D- and 2D-NMR-based metabolomics it was concluded that species included in group A (I. paraguariensis) were metabolically characterized by a higher amount of xanthines, and phenolics including phenylpropanoids and flavonoids; group B (I. dumosa var. dumosa and I. dumosa var. guaranina) with oleanane type saponins; group C (I. brasiliensis, I. integerrima, I. pseudobuxus and I. theezans) with arbutin and dicaffeoylquinic acids, and group D (I. argentina, I. brevicuspis, I. microdonta and I. taubertiana) with the highest level of ursane-type saponins. Clear metabolomic discrimination of Ilex species and varieties in this study makes the chemotaxonomic classification of Ilex species possible.     

The natural occurrence of Pandora heteropterae (Zygomycetes: Entomophthorales) infecting Lygus lineolaris (Hemiptera: Miridae)

An unknown fungal pathogen was recovered from Lygus lineolaris (Palisot de Beauvois) during a survey of parasitic and pathogenic natural enemies conducted in Franklin County, Arkansas. The pathogen was identified as Pandora heteropterae (Ba?azy) Keller based on characteristics of the morphology, as well as growth and sporulation on hosts. The fungus infected 11 of the 3405 (0.32%) wild L. lineolaris collected. In a laboratory host-range bioassay, five of seven hemipteran species from the families Miridae, Coreidae, Lygaeidae, and Pentatomidae were successfully infected. P. heteropterae was previously reported only once, from an unidentified host species in Poland. Here we describe the morphology and growth of P. heteropterae and discuss its potential impact on L. lineolaris in the field.     

Metabolic profiling and phylogenetic analysis of medicinal Zingiber species: Tools for authentication of ginger (Zingiber officinale Rosc)

Phylogenetic analysis and metabolic profiling were used to investigate the diversity of plant material within the ginger species and between ginger and closely related species in the genus Zingiber (Zingiberaceae). In addition, anti-inflammatory data were obtained for the investigated species. Phylogenetic analysis demonstrated that all Zingiber officinale samples from different geographical origins were genetically indistinguishable. In contrast, other Zingiber species were significantly divergent, allowing all species to be clearly distinguished using this analysis. In the metabolic profiling analysis, the Z. officinale samples derived from different origins showed no qualitative differences in major volatile compounds, although they did show some significant quantitative differences in non-volatile composition, particularly regarding the content of [6]-, [8]-, and [10]-gingerols, the most active anti-inflammatory components in this species. The differences in gingerol content were verified by HPLC. The metabolic profiles of other Zingiber species were very different, both qualitatively and quantitatively, when compared to Z. officinale and to each other. Comparative DNA sequence/chemotaxonomic phylogenetic trees showed that the chemical characters of the investigated species were able to generate essentially the same phylogenetic relationships as the DNA sequences. This supports the contention that chemical characters can be used effectively to identify relationships between plant species. Anti-inflammatory in vitro assays to evaluate the ability of all extracts from the Zingiber species examined to inhibit LPS-induced PGE(2) and TNF-alpha production suggested that bioactivity may not be easily predicted by either phylogenetic analysis or gross metabolic profiling. Therefore, identification and quantification of the actual bioactive compounds are required to guarantee the bioactivity of a particular Zingiber sample even after performing authentication by molecular and/or chemical markers.     

Flavonoids in the species of Cyrtomium (Dryopteridaceae) and related genera

Flavonoids in 19 Cyrtomium, three Cyrtogonellum and two Phanerophlebia taxa were surveyed. Major flavonoids were flavonol O-glycosides based on kaempferol, quercetin, and sometimes myricetin, and C-glycosylflavones, such as isovitexin, vitexin, isoorientin, orientin and their O-glycosides. The C-methylflavanones, farrerol and cyrtominetin, and their 7-O-glucosides were isolated from Cyrtomium devexiscapulae and Cyrtomium laetevirens. Flavanones have been reported from Cyrtomium falcatum sensu lato. Though C. falcatum sensu lato is divided into four taxa, i.e. C. falcatum subsp. falcatum, C. falcatum subsp. australe, C. falcatum subsp. littorale, and C. devexiscapulae, the occurrence of the flavanones was restricted to C. devexiscapulae, and they did not occur in C. falcatum sensu stricto.     

Hybridizations and genetic relationships among Lindernia species (Scrophulariaceae): L. procumbens and two subspecies of L. dubia

Lindernia procumbens and L. dubia are common annual weeds in flooded rice fields of Japan. Two subspecies of L. dubia, subsp. major and subsp. dubia, are usually recognized in Japan but they are both regarded as synonyms of L. dubia elsewhere. In a cluster analysis based on AFLP, most L. dubia subsp. major formed a separate cluster from L. dubia subsp. dubia although 11% of haplotypes classified using AFLP were not coincident with classification using the shape of leaf bases, which is the commonly used identification trait. Artificial F₁ plants between L. procumbens and L. dubia subsp. major, and between L. procumbens and L. dubia subsp. dubia did not produce seed. Forty percent of capsules produced by F₁ plants from these two subspecies were slimmer and 80% pollen were sterile in slimmer capsules. However, seed number of most F₁ capsules was not different from that of self-fertilized plants, suggesting that there was no complete reproductive isolation between the subspecies. Natural hybridization of these subspecies may have occurred but we are not aware of it because F₁ plants are rare and F₂ plants are indistinguishable from these subspecies.     

Flower colours and pigments in Disa hybrid (Orchidaceae)

Flower colours and the composition of pigments in the perianths of five cultivars of Disa orchids were analyzed. Carotenoids were major pigment components in the orange-red flowers of ‘Dawn Angel’. We identified two types of pigment composition in the red flowered cultivars: ‘San Francisco’ contained more carotenoids and less anthocyanins, while ‘Marlene’ contained more anthocyanins than carotenoids. The red-purple flowered cultivars, only contained slight amounts of carotenoids, and the red-purple colour was attributed to the relatively high density of a cyanidin-based anthocyanin. The importance of the characterization of pigments in the perianths of orchid has been discussed in both breeding for flower colour improvement and chemotaxonomy.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).