Quantification of cyanamide in young seedlings of Vicia species, Lens culinaris, and Robinia pseudo-acacia by gas chromatography-mass spectrometry

We quantified the cyanamide content of young leaves of nine Vicia species, Lens culinaris, and Robinia pseudo-acacia using a modified analytical procedure that made it possible to measure the cyanamide content of a single leaf. Recent molecular phylogenetic analysis suggests that cyanamide is present in V. benghalensis, which is placed in a monophyletic group with cyanamide-biosynthesizing plants, V. villosa and V. cracca; this suggestion was verified.     

Quantification of cyanamide contents in herbaceous plants

Cyanamide (NH2CN) is found in nature, although it has long been recognized as an industrial product. Distribution of cyanamide in the plant kingdom was investigated using a direct quantitative determination method to detect and measure cyanamide by stable isotope dilution gas chromatography-mass spectrometry (the SID-GC-MS method). The SID-GC-MS method proved to be a robust way to quantify cyanamide contents in the extracts of 101 species of herbaceous plants. The average recovery of cyanamide from all plants tested was 55.6+/-20.3%. Vicia villosa and V. cracca contained cyanamide at 369-498 microg/gFW and 3,460-3,579 microg/gFW respectively, while the other 99 species contained no detectable cyanamide (<1 microg/gFW). This result suggests that distribution of cyanamide in the plant kingdom is limited and uneven.     

Unravelling the phylogeny of tetraploid Vicia amoena (Fabaceae) and its diploid relatives using chromosomal landmarks

The chromosomal sites of 5S and 45S rDNA genes were revealed by fluorescence in situ hybridisation, and used as landmarks to resolve the ambiguous taxonomic status of the tetraploid Vicia amoena var. amoena (Fabaceae). Comparisons of the number and location of rDNA loci in V. amoena var. sericea, V. pseudorobus, V. dumetorum, V. pisiformis and V. cracca confirmed that V. amoena var sericea, or a closely related diploid, was a likely progenitor of the tetraploid. However, the tetraploid contains chromosomes bearing rDNA loci which are probably derived from another species, such as V. pseudorobus, suggesting that it is an allotetraploid and the product of an interspecific hybridisation event. This was corroborated by fluorescence in situ hybridisation with genomic DNA from V. amoena var sericea, which not only showed that the tetraploid contains more than one chromosome set, but also that its constituent sets have undergone considerable reorganisation.     

Quantification of Cyanamide Contents in Herbaceous Plants

Cyanamide (NH₂CN) is found in nature, although it has long been recognized as an industrial product. Distribution of cyanamide in the plant kingdom was investigated using a direct quantitative determination method to detect and measure cyanamide by stable isotope dilution gas chromatography–mass spectrometry (the SID–GC–MS method). The SID–GC–MS method proved to be a robust way to quantify cyanamide contents in the extracts of 101 species of herbaceous plants. The average recovery of cyanamide from all plants tested was 55.6±20.3%. Vicia villosa and V. cracca contained cyanamide at 369–498 μg/gFW and 3,460–3,579 μg/gFW respectively, while the other 99 species contained no detectable cyanamide (<1 μg/gFW). This result suggests that distribution of cyanamide in the plant kingdom is limited and uneven.     

Quantification of Cyanamide in Young Seedlings of Vicia Species,Lens culinaris,and Robinia pseudo-acacia by Gas Chromatography-Mass Spectrometry

We quantified the cyanamide content of young leaves of nine Vicia species, Lens culinaris, and Robinia pseudo-acacia using a modified analytical procedure that made it possible to measure the cyanamide content of a single leaf. Recent molecular phylogenetic analysis suggests that cyanamide is present in V. benghalensis, which is placed in a monophyletic group with cyanamide-biosynthesizing plants, V. villosa and V. cracca; this suggestion was verified.     

Vigna unguiculata is nodulated in Spain by endosymbionts of Genisteae legumes and by a new symbiovar (vignae) of the genus Bradyrhizobium

Vigna unguiculata was introduced into Europe from its distribution centre in Africa, and it is currently being cultivated in Mediterranean regions with adequate edapho-climatic conditions where the slow growing rhizobia nodulating this legume have not yet been studied. Previous studies based on rrs gene and ITS region analyses have shown that Bradyrhizobium yuanmingense and B. elkanii nodulated V. unguiculata in Africa, but these two species were not found in this study. Using the same phylogenetic markers it was shown that V. unguiculata, a legume from the tribe Phaseolae, was nodulated in Spain by two species of group I, B. cytisi and B. canariense, which are common endosymbionts of Genisteae in both Europe and Africa. These species have not been found to date in V. unguiculata nodules in its African distribution centres. All strains from Bradyrhizobium group I isolated in Spain belonged to the symbiovar genistearum, which is found at present only in Genisteae legumes in both Africa and Europe. V. unguiculata was also nodulated in Spain by a strain from Bradyrhizobium group II that belonged to a novel symbiovar (vignae). Some African V. unguiculata-nodulating strains also belonged to this proposed new symbiovar.     

Isozyme variation and phylogenetic relationships in Vicia subgenus Cracca (Fabaceae)

BACKGROUND AND AIMS: The phylogenetic relationships among 27 vetch species belonging to the subgenus Cracca of the genus Vicia were studied in comparison with three species of Lathyrus section Lathyrus on the basis of isozyme variation. METHODS: Isozymes encoded by 15 putative loci of ten enzymes were resolved by polyacrylamide gel electrophoresis and isozyme variation was analysed by using parsimony and neighbour-joining methods. KEY RESULTS: The analyses revealed 63 parsimony-informative and 36 species-specific orthozymes. Of the latter, 23 are monomophic and are suitable for identification of V. benghalensis, V. palaestina, V. dumetorum, V. pisiformis, V. sylvatica, V. onobrychioides, V. cappadocica, V. cretica, V. articulata, V. tetrasperma, V. ervilia, V. hirsuta and V. loiseleurii. Polymorphism with heterozygous and homozygous isozyme genotypes was found for V. cracca, V. tenuifolia, V. ochroleuca, V. villosa, V. sylvatica, V. cassubica, V. sparsiflora, V. megalotropis, V. altissima, V. onobrychioides, V. cassia, V. cretica and L. heterophyllus, reflecting outcrossing in these species. By contrast, V. benghalensis, V. palaestina, V. disperma, V. dumetorum, V. pisiformis, V. orobus, V. pauciflora, V. tetrasperma and V. loiseleurii had only homozygous isozyme genotypes at polymorphic loci. Isozyme-based phylogenetic trees are presented. CONCLUSIONS: Sections Cracca, Ervum, Pedunculatae and Lenticula of traditional taxonomy are monophyletic groups, whereas sections Oroboideae (= Vicilla) and Panduratae appear polyphyletic and section Cassubicae is split into two species-couples linked at a low level of support. Treatment of ervoid species in a separate subgenus Ervum is not supported because of its polyphyly.     

Ethno-ecological evidence for Hydnora abyssinica occurring in Johannesburg and Durban traditional medicine markets

Large quantities of plants are traded annually in South Africa's traditional medicine or ‘muthi’ markets. A resource in high demand in the Faraday (Johannesburg) and Warwick (Durban) markets is uMavumbuka, a root holoparasite usually identified as either Hydnora africana Thunb. or Sarcophyte sanguinea Sparrm. subsp. sanguinea. However, rhizomes regularly observed in Faraday between 1994 and 2008 did not resemble either species, thereby suggesting that a third, and undocumented, species was being harvested. This was confirmed when the rhizomes were identified as H. abyssinica A.Br. by an American parasitic plant expert. An ethno-ecological study was initiated to verify its occurrence in selected muthi markets. The study further aimed to investigate the distribution of H. abyssinica through trader interviews, host species localities and some previously misidentified herbarium specimens. The study revealed that H. abyssinica was the only uMavumbuka species present in Faraday and Warwick in 2009. Furthermore, the rhizomes were being harvested in KwaZulu-Natal—an area not previously known to be part of its distribution range. Re-evaluated herbarium vouchers and recent photographs taken in the Kruger National Park have confirmed that H. abyssinica occurs in KwaZulu-Natal, Limpopo, Mpumalanga, Gauteng and Swaziland and hence eastern southern Africa. Fragments of Acacia xanthophloea Benth. roots were identified on 93% of the samples that had host roots attached, and we suspect that H. abyssinica follows the distribution of A. xanthophloea in suitable habitats north from KwaZulu-Natal and adjoining the South African border with Swaziland and Mozambique. Acacia karroo Hayne and A. grandicornuta Gerstner have also been positively identified as host species in South Africa from herbarium records. Plant harvesters in the markets cited the common names of several other species that uMavumbuka “grows under” that may be identified as hosts to H. abyssinica in the future. The collection of specimens in areas identified by the harvesters and in areas of suitable habitat is important to verify the occurrence, distribution and habitat of H. abyssinica in eastern southern Africa.     

Cloning of casbene and neocembrene synthases from Euphorbiaceae plants and expression in Saccharomycescerevisiae

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.     

Quantitative real-time PCR detection of Pseudomonas oleovorans subsp. lubricantis using TaqMan-MGB assay in contaminated metalworking fluids

Metalworking fluids (MWFs) are highly prone to microbial contamination, which leads to their degradation and biofouling. Pseudomonas oleovorans subsp. lubricantis, a newly described subspecies, was found to be important to MWF fouling. However, the actual distribution of P. oleovorans subsp. lubricantis in MWF is difficult to study using standard culturing techniques. To overcome this, a study was conducted to design a specific quantitative real-time PCR (qPCR) assay using TaqMan^®MGB (minor groove binding) probe for its identification and estimated quantification in contaminated MWFs. The gyrB housekeeping gene sequence was selected for designing a TaqMan^® MGB primer-probe pair using the Allele ID^® 5.0 probe design software for the assay. Whole-cell qPCR was performed with MWF spiked directly with P. oleovorans subsp. lubricantis (eliminating DNA extractions using commercial kit); the primer-probe pair’s sensitivity was 10¹ colony forming units (CFU) ml⁻¹. The assay provided no amplification with other closely related Pseudomonas species found in MWFs indicating its specificity. It was successful in identifying and enumerating P. oleovorans subsp. lubricantis from several used MWFs having between 10⁴ and 10⁶ CFU ml⁻¹. The designed TaqMan^® MGB probe thus can be successfully used for the subspecies-specific identification of P. oleovorans subsp. lubricantis and facilitates the study of its impact on MWFs.     

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.     

Morphogenetic and diurnal variation of hypericin in some Hypericum species from Turkey during the course of ontogenesis

The genus Hypericum has received considerable interest from scientists, as it is a source of a variety of biologically active compounds including the hypericins. The present study was conducted to determine ontogenetic, morphogenetic and diurnal variation of the total hypericins content in some species of Hypericum growing in Turkey namely, Hypericum aviculariifolium subsp. depilatum var. depilatum (endemic), Hypericum perforatum and Hypericum pruinatum. The Hypericum plants were harvested from wild populations at vegetative, floral budding, full flowering, fresh fruiting and mature fruiting stages four times a day. Plants were dissected into stem, leaf and reproductive tissues, which were dried separately, and subsequently assayed for total hypericin content. The density of dark glands on leaves at full flowering plants was determined for each species. Floral parts had the highest hypericin content in all species tested. But diurnal fluctuation in the hypericin content of whole plant during the course of ontogenesis varied among the species. It reached the highest level at floral budding and tended to increase at night in H. aviculariifolium subsp. depilatum var. depilatum and H. pruinatum, whereas in H. perforatum hypericin content was the highest at full flowering and no diurnal fluctuation was observed. In general, hypericin content of leaves and whole plant was higher in H. aviculariifolium subsp. depilatum var. depilatum whose leaves had more numerous dark glands than those of the two other species.     

Molecular and phenotypic characterization of Vibrio aestuarianus subsp. francensis subsp. nov., a pathogen of the oyster Crassostrea gigas

Eleven Vibrio isolates invading the hemolymph of live and moribund oysters (Crassostrea gigas) collected in the field and from a hatchery in France, were characterized by a polyphasic approach. Phylogenetic analysis of 16S rRNA, gyrB and toxR genes indicated high homogeneity between these strains and the Vibrio aestuarianus type strain (ATCC35048(T)), and confirmed previous 16S rRNA analysis. In contrast, DNA:DNA hybridization was from 61% to 100%, while phenotypic characters and virulence tests showed a large diversity between the strains. Nevertheless, several common characters allowed the isolates to be distinguished from the reference strain. On the basis of several distinct phenotypic characteristics, it is proposed to establish two subspecies within the V. aestuarianus spp. group, V. aestuarianus subsp. aestuarianus [D. Tison, R. Seidler, Vibrio aestuarianus: a new species from estuarine waters and shellfish, Int. J. Syst. Bacteriol. (1983) 699-702] and V. aestuarianus subsp. francensis for these French isolates. The characters that differentiate the new strains from V. aestuarianus subsp. aestuarianus(T) are virulence (positive for 63% of the isolates) and 12:0 fatty acid content. The colonies were smaller and uncoloured, whereas no growth occurred at 35 degrees C or on TCBS, and the strains did not utilize several substrates, including L-serine, alpha-cyclodextrin, D-mannitol, alpha-glycyl-L-aspartic acid, L-threonine and glucose-1-phosphate.     

荆条叶性状对野外不同光环境的表型可塑性

光照是影响植物生长和分布的重要环境因子。对生长在野外5种不同光环境下(林外、阔叶林林缘、阔叶林林下、针叶林林窗和针叶林林下)的荆条的叶片进行取样研究,通过对光合作用光响应曲线、叶绿素荧光、叶绿素含量、叶片氮磷含量以及叶片形态的测量,来反映荆条对不同光环境的表型可塑性。研究结果表明,荆条叶片对于野外不同的光环境具有很好的适应机制,叶片功能性状受到结构性状的调节。低光下通过高的比叶面积(SLA)、单位质量叶绿素含量、光系统II最大量子产量,低的暗呼吸速率、光饱和点、光补偿点、叶绿素a,b的比值来提高对光能的利用效率,维持生长;高光下则通过与SLA有关的叶片结构的变化对光合作用进行调节。大多数的叶性状只受到日光照总量的影响,SLA的大小与日最高光强有关,可以对不同日变化模式的光照做出迅速的响应,是适应不同光照的敏感指标。尽管光照是不同光环境下影响荆条叶性状的主要环境因子,土壤养分含量同样会对叶性状产生影响,高土壤养分下的高叶长与叶柄长的比值体现了植物对资源获取和支撑结构之间分配的权衡。     

Allelopathy in the natural and agricultural ecosystems and isolation of potent allelochemicals from Velvet bean (Mucuna pruriens) and Hairy vetch (Vicia villosa).

We have studied on allelopathy of plants and developed methods to identify the effective substances in root exudates, leaf leacheate, and volatile chemicals emitted from plants. We found traditional cover plants that show allelopathic activity are useful for weed control. It could eliminate the use of synthetic chemicals for this purpose. Allelopathy is a natural power of plants to protect themselves by producing natural organic chemicals. Some endemic plants in Asia, already known by farmers in the region, as either cover crops used in intercropping, hedgerow, or agroforestry, were found to possess strong allelopathic abilities. Our group identified several allelochemicals from these plants. These allelopathic cover crops, mostly leguminous plants, provide protein rich food, and grow easily without artificial fertilizers, herbicides, insecticides and fungicides. In this regards, these allelopathic cover crops could save food shortage in rural area, and are useful for environmental conservation. Screenings of allelopathic plants by specific bioassays and field tests have been conducted. Hairy vetch (Vicia villosa) and Velvet bean (Mucuna pruriens) are two promising species for the practical application of allelopathy. An amino acid, L-DOPA, unusual in plants, plays an important role as allelochemical in Velvet bean (Mucuna pruriens). Hairy vetch is the most promising cover plant for the weed control in orchard, vegetable and rice production and even for landscape amendment in abandoned field in Japan. We have isolated "cyanamide", a well known nitrogen fertilizer, from Hairy vetch. This is the first finding of naturally produced cyanamide in the world.     

Glucosinolates in seeds of Neslia paniculata

The glucosinolate fraction in seeds of Neslia paniculata (L.) Desv., subsp. thracica (Velen.) Bornmüller, (Cruciferae), has been isolated and subjected to enzymic hydrolysis. The isothiocyanates thus produced have been separated and identified as cheirolin (II), (R)-9-methylsulfinylnonyl (III), (R)-10-methylsulfinyldecyl (IV), and (R)-11-methylsulfinylundecyl isothiocyanate (V), the last of which has not been previously encountered as a natural compound.     

Cyanogenesis in plants and arthropods

Cyanogenic glucosides are phytoanticipins known to be present in more than 2500 plant species. They are regarded as having an important role in plant defense against herbivores due to bitter taste and release of toxic hydrogen cyanide upon tissue disruption, but recent investigations demonstrate additional roles as storage compounds of reduced nitrogen and sugar that may be mobilized when demanded for use in primary metabolism. Some specialized herbivores, especially insects, preferentially feed on cyanogenic plants. Such herbivores have acquired the ability to metabolize cyanogenic glucosides or to sequester them for use in their own defense against predators. A few species of arthropods (within diplopods, chilopods and insects) are able to de novo biosynthesize cyanogenic glucosides and some are able to sequester cyanogenic glucosides from their food plant as well. This applies to larvae of Zygaena (Zygaenidae). The ratio and content of cyanogenic glucosides is tightly regulated in Zygaena filipendulae, and these compounds play several important roles in addition to defense in the life cycle of Zygaena. The transfer of a nuptial gift of cyanogenic glucosides during mating of Zygaena has been demonstrated as well as the involvement of hydrogen cyanide in male attraction and nitrogen metabolism. As more plant and arthropod species are examined, it is likely that cyanogenic glucosides are found to be more widespread than formerly thought and that cyanogenic glucosides are intricately involved in many key processes in the life cycle of plants and arthropods.     

Response of the entomopathogenic fungus Pandora neoaphidis to aphid-induced plant volatiles

We used a model plant-aphid system to investigate whether the aphid-specific entomopathogenic fungus Pandora neoaphidis responds to aphid-induced defence by the broad-bean plant, Vicia faba. Laboratory experiments indicated that neither in vivo sporulation, conidia size nor the in vitro growth of P. neoaphidis was affected by Acyrthosiphon pisum-induced V. faba volatiles. The proportion of conidia germinating on A. pisum feeding on previously damaged plants was significantly greater than on aphids feeding on undamaged plants, suggesting a direct functional effect of the plant volatiles on the fungus. However, there were no significant differences in the infectivity of P. neoaphidis towards A. pisum feeding on either undamaged V. faba plants or plants previously infested with A. pisum. Therefore, these results provide no evidence to suggest that P. neoaphidis contributes to plant indirect defence strategies.