Mechanism for the detoxification of aluminum in roots of tea plant (Camellia sinensis (L.) Kuntze)

To determine the mechanism of aluminum (Al) detoxification in the roots of tea plants (Camellia sinensis (L.) Kuntze), the amounts of Al and Al-chelating compounds (fluoride (F), organic acids and catechins) were measured and the chemical forms of Al in root cell extracts were identified by the application of 27Al-nuclear magnetic resonance (NMR) spectroscopy. Tea plants were cultivated in nutrient solutions containing 0, 4, 1.0 and 4.0 mM of Al at pH 4.2 for approximately 10 weeks. The levels of soluble Al, water-soluble oxalate and citrate, but not F, malate or catechins in young roots increased with an increase in the concentration of Al in the treatment solution. The 27Al NMR spectra of root tips and cell sap extracted from root tips that had been treated with Al were almost identical and had four signals, with two (11 and 16 ppm) apparently corresponding to the known chemical shifts of Al-oxalate complexes. In the spectra of cell sap, the resonances at 11 and 16 ppm increased with an increase in the Al contents. These results suggest that the levels of Al-oxalate complexes increased in response to an increase in the Al level, implying that oxalate is a key Al-chelating compound in the mechanism of Al detoxification in the tea root.     

Chemical forms of aluminum in xylem sap of tea plants (Camellia sinensis L.)

To identify the chemical forms of aluminum (Al) transported from roots to shoots of tea plants (C. sinensis L.), ²⁷Al-nuclear magnetic resonance and ¹⁹F NMR spectroscopy were used to analyze xylem sap.The concentration of Al in collected xylem sap was 0.29 mM, twice as high as that of F. Catechins were not detected in xylem sap. The concentration of malic acid in xylem sap was higher than that of citric acid, whereas the concentration of oxalic acid was negligible.There were two signals in the ²⁷Al NMR spectra of xylem sap, a larger signal at 11 ppm and a smaller one at −1.5 ppm. The former signal was consistent with the peak for an Al-citrate model solution, suggesting that an Al-citrate complex was present in xylem sap. Although the latter signal at −1.5 ppm was thought to indicate the presence of an Al-F complex (at 1.7 ppm) in xylem sap, there was only one signal at −122 ppm in the ¹⁹F NMR spectrum of xylem sap, indicating that the main F complex in xylem sap was F⁻.These results indicate that Al might be translocated as a complex with citrate, while Al-malate, Al-oxalate and Al-F complexes are not major Al complexes in xylem sap of tea plants.     

Metabolite profiling of alkaloids and strictosidine synthase activity in camptothecin producing plants

Camptothecin derivatives are clinically used anti-neoplastic alkaloids that biogenetically belong to monoterpenoid indole alkaloids. Camptothecin-related alkaloids from the methanol extracts of Ophiorrhiza pumila, Camptotheca acuminata and Nothapodytes foetida plants were profiled and identified using a reverse-phase high performance liquid chromatography coupled with on-line photodiode array detection and electrospray-ionization ion-trap mass spectrometry. A natural 10-glycosyloxy camptothecin, chaboside, was accumulated in tissues of O. pumila but not in C. acuminata and N. foetida. Anthraquinones regarded as phytoalexins were present in the extracts of hairy roots and calli but not in the differentiated plants of O. pumila. These findings demonstrated a remarkable difference in the constituents between the differentiated plants and the hairy roots or calli tissues. The activity of strictosidine synthase, a key enzyme of camptothecin biosynthesis, was detected in the protein extracts of stems and roots of O. pumila, being correlated with the pattern of strictosidine synthase mRNA expression.     

Clonal variation of tea [Camellia sinensis (L.) O. Kuntze] in countering water deficiency

Various clones of tea [Camellia sinensis (L.) O. Kuntze] such as TTL-1, TTL-2, TTL-4, TTL-5, TTL-6, UPASI-2 and UPASI-3 planted in the field were subjected to soil moisture stress conditions by withholding irrigation. A control set of the same clones were maintained by watering regularly. The soil water content of the irrigated and non irrigated plants was monitored through the soil moisture status. The extent of effect of drought on tea plants were monitored through various physiological parameters such as shoot weight, leaf water potential, chlorophyll and carotenoid content, chlorophyll fluorescence (Fv/Fm), net photosynthetic rate, transpiration rate, stomatal conductance and biochemical parameters such as extent of proline accumulation and free radical generation. These parameters were studied on the 30 d of non irrigation and on the 5 d during recovery from drought. The plants recovered when re-irrigated after 30 d of non-irrigation, which suggests that permanent wilting did not occur due to non-irrigation up to 30 d. On the 30 d of non-irrigation the clones TTL-1, TTL-6 and UPASI-2 showed lesser reduction of shoot weight, leaf water potential, chlorophyll fluorescence, photosynthetic rate, transpiration rate and stomatal conductance and increased proline and lesser lipid peroxidation as compared to the other clones. From these results it can be concluded that the clones TTL-1, TTL-6 and UPASI-2 are comparatively more drought tolerant than the clones TTL-2, TTL-4, TTL-5 and UPASI-3.     

Molecular characterization and enzymatic activity of laccases in two Pleurotus spp. with different pathogenic behaviour

Pleurotus eryngii and P. ferulae, two species belonging to the P. eryngii complex, synthesize laccases, ligninolytic enzymes that play a role in the host-pathogen interaction in the first step of infection. Ecological studies have shown that although both fungi have been recognized as saprophytes, P. eryngii weakly pathogenic when colonizing the roots and stems of Eryngium campestre, whereas P. ferulae is mostly pathogenic to Ferula communis. The paper describes the genomic organization of four putative laccase genes (lac1, lac2, lac3, and lac5-like gene; gene names were assigned on the basis of sequence homologies) of P. eryngii and P. ferulae. The mRNA expression and enzymatic activity of the laccases were analysed under culture conditions where a source of lignin (wheat bran) or lyophilized roots of E. campestre or F. communis were present. These experiments indicated that the four lac-like genes were differentially regulated in the two mushrooms. Specifically, the addition of the lyophilized roots of the respective host plant to the culture media induced an advance in the mRNA expression of the four lac-like genes and a seven-fold higher total laccase activity in P. ferulae than in P. eryngii. The results obtained are discussed in relation to the possible role of laccases in the interaction of P. eryngii and P. ferulae with their respective host.     

Metabolite profiling and quantification of phenolic compounds in methanol extracts of tomato fruit

The consumption of tomatoes and tomato products has been associated with a reduction in the risk of contracting some types of cancer and other chronic diseases. These beneficial properties may be attributed to the presence of key metabolites and the interactions among them. We have developed and validated an analytical method for the comprehensive profiling of semi-polar metabolites in the methanol extract of three cultivars of raw tomatoes (Daniela, Raf and Rambo) grown in Almería, in south-east Spain. Diode-array and time-of-flight/ion-trap mass spectrometry detectors were used to ensure the wide detection of metabolites with highly divergent properties. The masses thus detected were assigned by matching their accurate mass-signals with tomato compounds reported in the literature, and supplemented by UV and MS/MS information, reference compounds and existing metabolite databases. In this way we were able to identify tentatively 135 compounds belonging to various structural classes, 21 of which are to our knowledge reported for the first time in the tomato fruit. Among the metabolites identified, the most abundant were phenolic compounds. This class of secondary metabolites is attracting considerable attention from producers and consumers due to their antioxidant activity and nutritional properties. Their quantitative analysis was achieved by using closely related derivatives for each family.     

Identification of Vitis vinifera (-)-alpha-terpineol synthase by in silico screening of full-length cDNA ESTs and functional characterization of recombinant terpene synthase

The flavour and aroma of certain Vitis vinifera grape varieties is dominated by volatile terpenes and small volatile aldehydes. Monoterpenes contribute to the final grape and wine aroma and flavour in form of free volatiles and as glycoside conjugates of monoterpene alcohols. Typical monoterpenol components of the cultivar Gewürztraminer and other aroma-rich grape varieties are linalool, geraniol, nerol, citronellol, and alpha-terpineol. In a functional genomics effort to identify genes for the formation of monoterpene alcohols in V. vinifera, a database of full-length cDNA sequences was screened in silico and yielded two clones for putative monoterpene synthases. The gene products were functionally characterized by expression in Escherichia coli, in vitro enzyme assay and gas chromatography-mass spectrometry (GC-MS) product identification as multi-product (-)-alpha-terpineol synthases.     

Nosema chrysorrhoeae n. sp. (Microsporidia), isolated from browntail moth (Euproctis chrysorrhoea L.) (Lepidoptera, Lymantriidae) in Bulgaria: characterization and phylogenetic relationships

A new microsporidian parasite Nosema chrysorrhoeae n. sp., isolated in Bulgaria from the browntail moth (Euproctis chrysorrhoea L.), is described. Its life cycle includes two sequential developmental cycles that are similar to the general developmental cycles of the Nosema-like microsporidia and are indistinguishable from those of two Nosema spp. from Lymantria dispar. The primary cycle takes place in the midgut tissues and produces binucleate primary spores. The secondary developmental cycle takes place exclusively in the silk glands and produces binucleate environmental spores. N. chrysorrhoeae is specific to the browntail moth. Phylogenetic analysis based on the ssu rRNA gene sequence places N. chrysorrhoeae in the Nosema/Vairimorpha clade, with the microsporidia from lymantriid and hymenopteran hosts. Partial sequences of the lsu rRNA gene and ITS of related species Nosema kovacevici (Purrini K., Weiser J., 1975. Natürliche Feinde des Goldafters, Euproctis chrysorrhoea L., im Gebiet von Kosovo, FSR Jugoslawien. Anzeiger fuer Sch?dlingskunde, Pflanzen-Umweltschutz, 48, 11-12), Nosema serbica Weiser, 1963 and Nosema sp. from Lymantria monacha was obtained and compared with N. chrysorrhoeae. The molecular data indicate the necessity of future taxonomic reevaluation of the genera Nosema and Vairimorpha.     

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia

Nosema ceranae is a recently described pathogen of Apis mellifera and Apis cerana. Relatively little is known about the distribution or prevalence of N. ceranae in the United States. To determine the prevalence and potential impact of this new pathogen on honey bee colonies in Virginia, over 300 hives were sampled across the state. The samples were analyzed microscopically for Nosema spores and for the presence of the pathogen using real-time PCR. Our studies indicate that N. ceranae is the dominant species in Virginia with an estimated 69.3% of hives infected. Nosema apis infections were only observed at very low levels (2.7%), and occurred only as co-infections with N. ceranae. Traditional diagnoses based on spore counts alone do not provide an accurate indication of colony infections. We found that 51.1% of colonies that did not have spores present in the sample were infected with N. ceranae when analyzed by real-time PCR. In hives that tested positive for N. ceranae, average C_T values were used to diagnose a hive as having a low, moderate, or a heavy infection intensity. Most infected colonies had low-level infections (73%), but 11% of colonies had high levels of infection and 16% had moderate level infections. The prevalence and mean levels of infection were similar in different regions of the state.     

Biological and molecular characterization of a multicapsid nucleopolyhedrovirus from Thysanoplusia orichalcea (L.) (Lepidoptera: Noctuidae)

A multicapsid nucleopolyhedrovirus (ThorMNPV) that was co-isolated with a single nucleocapid ThorSNPV from mixed infected larvae of Thysanoplusia orichalcea L. (Lepidoptea: Noctuidae) is characterized. Scanning electron microscopy of ThorMNPV showed a dodecahedral-shaped occlusion body (OB). The occluded virions contained one to as many as eight nucleocapsids/virion. Virion band profiles in gradient centrifugation were consistent in at least 10 rounds of centrifugation from different virion sample preparations. The ThorMNPV had high virulence to third instar Trichoplusia ni and Pseudoplusia includens with LD50 values of 17 and 242OBs per larva, respectively. However, ThorMNPV did not cause mortality in Spodoptera exigua, Spodoptera frugiperda, Spodoptera eridania, Anticarsia gemmatalis, and Helicoverpa zea. ThorMNPV replicates in cells of various tissues such as the fat body and tracheal epithelium cells. T. ni High 5 cells were permissive to ThorMNPV in terms of infection and viral DNA transfection, but SF-21 was less permissive and the infection process was slower. Production of OBs by ThorMNPV in the nuclei of SF-21 was not well pronounced. The genome size of ThorMNPV was estimated to be 136 kb. The polyhedrin gene open reading frame (ORF) was cloned and completely sequenced. The promoter sequence is identical to that of Autographa californica MNPV. Phylogenetic analyses using partial sequences of the polh, lef-8, and lef-9 revealed that ThorMNPV is a member of the Group I NPVs and is related but distinct from the AcMNPV/Rachiplusia ou NPV/Bombyx mori NPV cluster.     

Expression cloning screening of a unique and full-length set of cDNA clones is an efficient method for identifying genes involved in Xenopus neurogenesis

Functional screens, where a large numbers of cDNA clones are assayed for certain biological activity, are a useful tool in elucidating gene function. In Xenopus, gain of function screens are performed by pool screening, whereby RNA transcribed in vitro from groups of cDNA clones, ranging from thousands to a hundred, are injected into early embryos. Once an activity is detected in a pool, the active clone is identified by sib-selection. Such screens are intrinsically biased towards potent genes, whose RNA is active at low quantities. To improve the sensitivity and efficiency of a gain of function screen we have bioinformatically processed an arrayed and EST sequenced set of 100,000 gastrula and neurula cDNA clones, to create a unique and full-length set of approximately 2500 clones. Reducing the redundancy and excluding truncated clones from the starting clone set reduced the total number of clones to be screened, in turn allowing us to reduce the pool size to just eight clones per pool. We report that the efficiency of screening this clone set is five-fold higher compared to a redundant set derived from the same libraries. We have screened 960 cDNA clones from this set, for genes that are involved in neurogenesis. We describe the overexpression phenotypes of 18 single clones, the majority of which show a previously uncharacterised phenotype and some of which are completely novel. In situ hybridisation analysis shows that a large number of these genes are specifically expressed in neural tissue. These results demonstrate the effectiveness of a unique full-length set of cDNA clones for uncovering players in a developmental pathway.     

Effects of fungal culture filtrates of Verticillium lecanii (Lecanicillium spp.) hybrid strains on Heterodera glycines eggs and juveniles

Many nematode-antagonistic fungi produce secondary metabolites and enzymes that demonstrate toxicity against plant-parasitic nematodes. The objective of this study was to evaluate the effects of fungal culture filtrates of Verticillium lecanii hybrid strains on mature eggs, embryonated eggs (eggs fertilized but without development of juveniles), and second-stage juveniles (J2) of Heterodera glycines and to compare these effects with those of their parental strains. The fungal culture filtrates of certain hybrid strains inhibited egg hatch of mature eggs. Furthermore, the fungal culture filtrates of two hybrid strains, AaF23 and AaF42, exhibited high toxicity against embryonated eggs of H. glycines. However, most of the fungal culture filtrates of V. lecanii did not inactivate J2. These results suggested that enzymes or other active compounds produced by the fungal culture filtrates of V. lecanii exhibit activity against specific stages in the H. glycines life cycle. In addition, based on a visual assessment of the morphological changes in eggs caused by filtrates of each strain, there were differences between the hybrid strains and their respective parental strains with regard to the active substances produced by V. lecanii against the embryonated eggs. As a result of promoting recombination of whole genomes via protoplast fusion, several hybrid strains may have enhanced production of active substances that are different from those produced by their parental strains. It was concluded that natural substances produced by V. lecanii are one of the important factors involved in the suppression of H. glycines damage.     

Genetic evidence for three discrete taxa of Melampsora (Pucciniales) affecting willows (Salix spp.) in New York State

Rust fungi in the genus Melampsora (Pucciniales) are the most important pathogens of shrub willows (Salix spp.) cultivated for biomass in New York State and temperate regions worldwide. The taxonomy and species identification of these fungi historically have been problematic as they are morphologically indistinguishable on willow and often have complex life histories. Melampsora of Salix in North America, therefore, have been circumscribed to the collective species Melampsora epitea Thüm. and further delineated to formae speciales by aecial host. Ribosomal DNA (rDNA) data was obtained from 75 collections/isolates of Melampsora in NY State affecting either native and cultivated Salix spp. or suspected alternate hosts. Maximum likelihood (ML), maximum parsimony (MP), and Bayesian (BI) analyses were conducted on three data partitions (individual and concatenated): complete internal transcribed spacer (ITS) and partial large subunit (LSU) rDNA sequences for all collections. Analyses of the ITS and concatenated ITS-LSU sequences revealed that Melampsora on native and cultivated willows in NY State consisted of three phylogenetically delineable taxa (phylotaxa); monophyly for each phylotaxon was strongly supported by ML, MP, and BI credibility values. Phylotaxa were also delimited phylogenetically by aecial host: Alpine currant (Ribes alpinum), eastern larch (Larix laricina), or balsam fir (Abies balsamea).     

Isolation and characterization of visceral excitatory neuropeptides from striped mullet (Mugil cephalus L.) brain

From a brain extract of the catadromous fish, striped mullet (Mugil cephalus), two visceral excitatory neuropeptides (Mvp-1 and Mvp-2) were isolated by means of reversed phase chromatography together with bioassay on fish hindgut. The primary structure of Mvp-1 was elucidated to be SGPAGVLamide by ESI-Q-TOF mass spectrometry. The threshold concentration of Mvp-1 that changes spontaneous contraction of mullet hindgut was between 10(-9) and 10(-8) M. In addition, Mvp-1 was found to exert excitatory activities on some other smooth muscle segments (oviduct and esophagus) of mullet but it did not show any effect on body wall muscle strips. Therefore, the present study suggests that Mvp-1 and Mvp-2 peptides act as factors that control visceral contractions of mullet gastrointestinal tract.     

New genotypes and factors associated with Cryptosporidium detection in mussels (Mytilus spp.) along the California coast

A 3 year study was conducted to evaluate mussels as bioindicators of faecal contamination in coastal ecosystems of California. Haemolymph samples from 4680 mussels (Mytilus spp.) were tested for Cryptosporidium genotypes using PCR amplification and DNA sequence analysis. Our hypotheses were that mussels collected from sites near livestock runoff or human sewage outflow would be more likely to contain the faecal pathogen Cryptosporidium than mussels collected distant to these sites, and that the prevalence would be greatest during the wet season when runoff into the nearshore marine environment was highest. To test these hypotheses, 156 batches of sentinel mussels were collected quarterly at nearshore marine sites considered at higher risk for exposure to livestock runoff, higher risk for exposure to human sewage, or lower risk for exposure to both faecal sources. Cryptosporidium genotypes detected in Haemolymph samples from individual mussels included Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium andersoni, and two novel Cryptosporidium spp. Factors significantly associated with detection of Cryptosporidium spp. in mussel batches were exposure to freshwater outflow and mussel collection within a week following a precipitation event. Detection of Cryptosporidium spp. was not associated with higher or lower risk status for exposure to livestock faeces or human sewage sources. This study showed that mussels can be used to monitor water quality in California and suggests that humans and animals ingesting faecal-contaminated water and shellfish may be exposed to both host-specific and anthropozoonotic Cryptosporidium genotypes of public health significance.