Genetic Diversity and Structure of Dalmatian Pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip., Asteraceae) within the Balkan Refugium

Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip.) is an outcrossing, perennial insecticidal plant, restricted to the eastern Adriatic coast (Mediterranean). Amplified fragment-length polymorphisms (AFLP) were used to investigate the genetic diversity and structure within and among 20 natural plant populations. The highest level of gene diversity, the number of private alleles and the frequency down-weighted marker values (DW) were found in northern Adriatic populations and gradually decreased towards the southern boundary of the species range. Genetic impoverishment of these southern populations is most likely the result of human-related activities. An analysis of molecular variance (AMOVA) indicated that most of the genetic diversity was attributed to differences among individuals within populations (85.78%), which are expected due to the outcrossing nature of the species. A Bayesian analysis of the population structure identified two dominant genetic clusters. A spatial analysis of the genetic diversity indicated that 5.6% of the genetic differentiation resulted from isolation by distance (IBD), while 12.3% of the genetic differentiation among populations followed the pattern of isolation by environmental distance (IBED). Knowledge of the genetic diversity patterns of the natural populations and the mechanism behind these patterns is required for the exploitation and possible conservation management of this endemic and economically important species.     

Chemical and genetic characterization of calli derived from somatic hybridization between tansy (Tanacetum vulgare L.) and pyrethrum (Tanacetum cinerariifolium (Trevir.) Schultz-Bip.)

 Pyrethrum (Tanacetum cinerariifolium (Trevir.) Schultz-Bip.) produces environmentally benign pesticides, the pyrethrins, and tansy (Tanacetum vulgare L.) lower terpenes of variable biological effectiveness. As an approach to improve the oil content and composition of tansy for enhanced biological activity, a somatic hybridization technique between tansy and pyrethrum was established. About 1×10⁶ of leaf-mesophyll protoplasts of both species were mixed and fused with a solution containing 15% polyethylene glycol. Light-green and yellowish calli developed from the fusion experiments. The fusion-derived calli grew vigorously on MS medium supplemented with 6.4 mg l^-1 of BAP, 0.8 mg l^-1 of NAA, and 30–40 g l^-1 of glucose. Nuclear DNA content, RAPD patterns, and volatile compounds were analyzed to determine the hybridity of the calli. The nuclear DNA content of the tansy and pyrethrum genotypes, and the protoplast-derived calli of tansy were 6.41, 7.39, 13.84, and 8.11 pg, respectively. The nuclear DNA content of individual calli derived from the protoplast fusion between tansy +tansy ranged from 8.84 (F43A) to 19.59 pg (F43C) while those of the tansy+pyrethrum fusions were 10.66 (F46A) and 31.87 pg (F46B). Using four 10-mer primers a total of 56 RAPD-PCR fragments were produced. The distance matrices of fragments were calculated by average linkage cluster analysis. Two visually separated clusters were observed. One cluster consisted of the two tansy genotypes and the fusion-derived callus F43A; the other consisted of pyrethrum and fusion-derived calli F46B and F46C. Volatile compounds, such as decadienal, artedouglasia oxide, heptadecane, syringaldehyde and coniferyl alcohol, analyzed by gas chromatography mass spectrometry, were found only in the protoplast fusion-derived calli F43A and F46B. Several less volatile compounds were also detected only in fusion calli. Hexadecanoic and linoleic acids were common to fusion-derived calli and tansy, and one unknown compound to fusion-derived calli and pyrethrum. Pyrethrins I and II were detected from pyrethrum, but not from the fusion-derived calli. The additive nuclear DNA content of protoplast fusion-derived calli and the results of the RAPDs suggest that interspecific fusions had occurred. The small number of volatile compounds detected from both the fusion calli and from the donor species indicates that the unorganized callus tissue is unable to produce tissue-specific volatile compounds. Received: 4 August 1998 / Accepted: 30 September 1998     

Pyrethrin from Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.): biosynthesis,biological activity,methods of extraction and determination

Phytochemistry Reviews - Pyrethrin is a potent biopesticide, a natural mixture of six compounds (pyrethrin I and II, cinerin I and II, and jasmolin I and II), biosynthesized in plants of Dalmatian...     

Correction to: Pyrethrin from Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.): biosynthesis,biological activity,methods of extraction and determination

Phytochemistry Reviews -     

Requirement of Catalytic-Triad and Related Amino Acids for the Acyltransferase Activity of Tanacetum cinerariifolium GDSL Lipase/Esterase TcGLIP for Ester-Bond Formation in Pyrethrin Biosynthesis

We have recently discovered that a GDSL lipase/esterase (TcGLIP) in Tanacetum cinerariifolium catalyzed acyltransferase activity to form an ester bond in the natural insecticide, pyrethrin. TcGLIP contained Ser40 in Block I, Gly64 in Block II, Asn168 in Block III and Asp318 and His321 in Block V, suggesting underlying hydrolase activity, although little is known about their role in acyltransferase activity. We expressed TcGLIP here in Esherichia coli as a fusion with maltose-binding protein (MBP), part of the fusion being cleaved with a protease to obtain MBP-free TcGLIP. A kinetic analysis revealed that the MBP moiety scarcely influenced the kinetic parameters. The effects on acyltransferase activity of mutations of Gly64, Asn168, Asp318 and His321 were investigated by using MBP-fused TcGLIP. Mutations of these amino acids markedly reduced the acyltransferase activity, suggesting their critical role in the production of pyrethrins.     

除虫菊内生真菌的分离及其发酵产物抑菌活性筛选

从除虫菊(Pyrethrumcinerariifolium Trev.)的根、茎、叶和花中分离出128株内生真菌。生长速率法测定结果表明,有56株内生真菌发酵液10倍稀释液至少对供试的番茄灰霉病菌(Botrytis cinerea)、辣椒疫霉病菌(Phytophthora capsici)、苹果炭疽病菌(Colletotrichum gloeosporioides)、玉米大斑病菌(Exserohilumturcicum)、小麦纹枯病菌(Rhizoctonia cerealis)和小麦赤霉病菌(Fusarium graminearum)等6种病原菌中的一种抑菌活性达75%以上;活体组织法测定结果,Y1、Y2、H2共3株菌的发酵液对番茄灰霉病的防效达到50%以上;盆栽试验结果表明,Y1、Y2、Y7、H2、G13共5株菌株发酵液对辣椒疫霉病的防治效果达到50%以上。综合分析认为,Y1、Y2、H2、G13等内生真菌的抑菌作用值得进一步研究。     

Chemotype Diversity of Indigenous Dalmatian Sage (Salvia officinalis L.) Populations in Montenegro

To identify how many chemotypes of Salvia officinalis exist in Montenegro, the chemical composition of the essential oils of 12 wild‐growing populations was determined by GC‐FID and GC/MS analyses. Among the 40 identified constituents, the most abundant were cis‐thujone (16.98–40.35%), camphor (12.75–35.37%), 1,8‐cineol (6.40–12.06%), trans‐thujone (1.5–10.35%), camphene (2.26–9.97%), borneol (0.97–8.81%), viridiflorol (3.46–7.8%), limonene (1.8–6.47%), α‐pinene (1.59–5.46%), and α‐humulene (1.77–5.02%). The composition of the essential oils under study did not meet the ISO 9909 requirements, while the oils of populations P02–P04, P09, and P10 complied with the German Drug Codex. A few of the main essential‐oil constituents appeared to be highly intercorrelated. Strong positive correlations were observed between α‐pinene and camphene, camphene and camphor, as well as between cis‐thujone and trans‐thujone. Strong negative correlations were evidenced between cis‐thujone and α‐pinene, cis‐thujone and champhene, cis‐thujone and camphor, as well as between trans‐thujone and camphene. Multivariate analyses allowed the grouping of the populations into three distinct chemotypes, i.e., Chemotype A, rich in total thujones, Chemotype B, with intermediate contents of thujones, α‐pinene, camphene, and camphor and high borneol contents, and Chemotype C, rich in camphor, camphene, and α‐pinene. The chemotypes did not significantly differ in the total essential‐oil content and the cis/trans‐thujone ratio.     

Trypanosoma cruzi: antiprotozoal activity of parthenolide obtained from Tanacetum parthenium (L.) Schultz Bip. (Asteraceae, Compositae) against epimastigote and amastigote forms

This study reports the activity of crude extracts, fractions and parthenolide (pure compound) obtained from Tanacetum parthenium against two forms of the parasite Trypanosoma cruzi. Feverfew is a traditional herbal medicine that has been used for the treatment of migraine, fever and arthritis. Activity against epimastigote forms was observed for crude extracts, fractions and parthenolide, and a progressive increase in the antitrypanosomal effect was observed in the course of the purification process. The pure compound showed IC50/96h and IC90/96h of 0.5 microg/ml and 1.25 microg/ml, respectively. The cytotoxic effect of parthenolide in LLMCK2 cells was 3.2 microg/ml (CC50/96h) and the selectivity index was 6.4. No hemolysis was detected for the pure compound. The internalization index of T. cruzi in LLMCK2 cells was reduced almost 51% at the concentration of 2 microg/ml of parthenolide, and 96.6% at 4 microg/ml. Scanning and transmission electron microscopy permitted observation of morphological modifications and ultrastructural alterations.     

Genetic Diversity in Natural Populations of Arabidopsis thaliana (L.) Heynh. from Karelia

The genetic structure of ten natural populations of Arabidopsis thaliana (L.) Heynh. at eight isozyme loci was studied. The populations were located in the northern part of the species range, 200 km from the north to the south along the Onega Lake coast in Karelia. Considerable genetic diversity (P _99% = 43.7, H _obs = 0.003) was revealed that is not typical of populations of self-pollinating plant species. A direct correlation between the proportion of polymorphic loci and geographical latitude was shown (r = 0.68; P < 0.05). It is suggested that a high polymorphism level in Karelian Arabidopsis thaliana (L.) populations increasing from the south to the north is due to extreme environmental conditions in the northern part of the species range. The distribution of genetic diversity within and between populations is typical of self-pollinating species: the larger part of the total diversity resides among populations (G _ST = 0.583).     

High Diversity of Indigenous Populations of Dalmatian Sage (Salvia officinalis L.) in Essential‐Oil Composition

Essential oils of 25 indigenous populations of Dalmatian sage (Salvia officinalis L.) that represent nearly half of native distribution area of the species were analyzed. Plantlets collected from wild populations were grown in the same field under the same environmental conditions and then sampled for essential‐oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Among the 62 compounds detected, eight (cis‐thujone, camphor, trans‐thujone, 1,8‐cineole, β‐pinene, camphene, borneol, and bornyl acetate) formed 78.13–87.33% of essential oils of individual populations. Strong positive correlations were observed between camphor and β‐pinene, β‐pinene and borneol, as well as between borneol and bornyl acetate. The strongest negative correlation was detected between camphor and trans‐thujone. Principal component analysis (PCA) on the basis of eight main compounds showed that first main component separated populations with high thujone content, from those rich in camphor, while the second component separated populations rich in cis‐thujone from those rich in trans‐thujone. Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis‐thujone; trans‐tujone, and camphor/β‐pinene/borneol/bornyl acetate. We propose that differences in essential oils of 25 populations are mostly genetically controlled, since potential environmental factors were controlled in this study.     

Pollen morphology of endemic species of the Gonospermum Less., Lugoa DC. and Tanacetum L. complex (Asteraceae: Anthemideae) in the Canary Islands (Spain), and its taxonomical implications

The pollen of nine endemic taxa of the Gonospermum Less., Lugoa DC. and Tanacetum L. complex and of T. parthenium L. (introduced species from the Canary Islands) was studied using light and scanning electron microscopy with the aim of extending the knowledge of their morphology and to investigate whether pollen morphology may help clarifying the controversial taxonomy of these taxa. Twenty six populations in the Canary Islands (Spain) were studied and the following parameters were measured: polar axis (P), equatorial diameter (E), polar exine thickness (Ex1), length (L) and thickness (A) of the spines in the meridional optical section, exine thickness at the centre of the mesocolpium in the interapertural area (Ex2) and distance between spines (De) in the equatorial optical section. The studies show that the pollen of the three genera is very similar regarding shape, apertures, and ornamentation, but may be distinguished by size and exine thickness.     

Effects of juvenile hormone I, precocene I and precocene II on the progeny of Microplitis rufiventris Kok. female when administered via its host, Spodoptera littoralis (Boisd.)

Reproductive biologies of Microplitis rufiventris Kok. females resulting from topically treated Spodoptera littoralis (Boisd.) larvae with constant effective doses of juvenile hormone I (JHI, 1 μg), precocene I (PI, 25 μg) or PII (25 μg) were investigated. Although the female wasps were treated during their presence as eggs or larvae in their hosts, the complete effects of the test compound were not apparent until the wasps had become adults. On the bases of the obtained results, the reproductive inhibition activity caused by the test compounds comprises of two categories: (1) reduction in progeny production, and (2) induction of significant proportion of imperfect ‘non‐functional’ parasitoid progeny. Whereas, the adverse effect of JHI is only restricted to the second category, the adverse effects of PI or PII fall into both categories. Thus, workers should be aware of the delayed effects of new generations of pesticides which may occur in later stages of the non‐target insects.     

Morphometric and Microsatellite Diversity in Seabuckthorn (Hippophae rhamnoides L.) Natural Populations Originating from the Different Geographical Regions of the Indian Himalayas

Plant Molecular Biology Reporter - Seabuckthorn (Hippophae rhamnoides L.), an upcoming superfood plant, has attracted researchers’ attention worldwide for its medicinal, nutritional, and...     

High Genetic Diversity and Differentiation in Relict Lowland Populations of Gentianella austriaca (A. and J. Kern.) Holub (Gentianaceae)

Abstract: The biennial Gentianella austriaca (A. & J. Kern.) Holub, representing a nutrient-poor grassland taxon of low competition power, is becoming rare in the lowlands of eastern Austria due to changes in land use. To estimate effects of isolation and decreasing population sizes, as well as evolutionary relationships, we investigated variation in isozymes and morphological characters within and between seven populations from the mountains, foothills, and lowlands. Additionally, data on reproduction, habitat, germination and population sizes were collected to examine possible causes of variation and differentiation. We found highest genetic diversity (v_a, v_go) in the lowland and foothill populations, and highest genetic differentiation (D_ja, D_jgo) (i.e., lowest genetic identity: Nei's I) in the lowland populations. The low diversity of the mountain populations might indicate that they are derived from lowland populations. Surprisingly, highest genetic diversity was found in the smallest population. This indicates that in small remnant populations of taxa with a mixed mating system, genetic diversity might be maintained even after many generations after reducing population size dramatically. We found some relationship between genetic diversity and high fitness (germination success) and (inversely) with seed size. Plant size and reproductive success are negatively correlated with altitude, whereas flower size and seed size seem to be subject to other forces of selection. Combining all morphometric, reproductive and genetic traits, the lowland populations are most strongly differentiated and therefore of highest conservation priority.     

First Report on Rare Unifloral Honey of Endemic Moltkia petraea (Tratt.) Griseb. from Croatia: Detailed Chemical Screening and Antioxidant Capacity

Rare Moltkia petraea (Tratt .) Griseb . honey from Croatia was first time characterised. The spectrophotometric assays on CIE L*a*b*C_ab*h_ab° colour coordinates, total phenol content and antioxidant capacity (FRAP , CUPRAC , DPPH ^? and ABTS ^?+ assays) determined higher honey values generally close to dark honeys ranges. Headspace solid‐phase microextraction (HS ‐SPME ) on two fibres after GC ‐FID and GC /MS revealed the major compounds 2‐phenylacetaldehyde (12.8%; 15.6%), benzaldehyde (11.1%; 10.0%), octane (9.3%; 7.6%), nonane, propan‐2‐one, pentan‐2‐one, pentanal and nonanal (4.9%; 14.5%). Ultrasonic solvent extraction (USE ) mainly isolated non‐specific higher molecular compounds characteristic of the comb environment. Targeted HLPC ‐DAD analysis of the honey determined higher concentration of phenylalanine (212.08 mg/kg) and lumichrome (16.25 mg/kg) along with tyrosine and kojic acid. The headspace composition (chemical fingerprint) and high concentration of lumichrome can be considered particular for M . petraea honey.     

Variation of the Chemical Composition and Antimicrobial Activity of the Essential Oils of Natural Populations of Tunisian Daucus carota L. (Apiaceae)

The essential oils of Daucus carota L. (Apiaceae) seeds sampled from ten wild populations spread over northern Tunisia were characterized by GC‐FID and GC/MS analyses. In total, 36 compounds were identified in the D. carota seed essential oils, with a predominance of sesquiterpene hydrocarbons in most samples (22.63–89.93% of the total oil composition). The main volatile compounds identified were β‐bisabolene (mean content of 39.33%), sabinene (8.53%), geranyl acetate (7.12%), and elemicin (6.26%). The volatile composition varied significantly across the populations, even for oils of populations harvested in similar areas. The chemometric principal component analysis and the hierarchical clustering identified four groups, each corresponding to a composition‐specific chemotype. The in vitro antimicrobial activity of the isolated essential oils was preliminarily evaluated, using the disk‐diffusion method, against one Gram‐positive (Staphylococcus aureus) and two Gram‐negative bacteria (Escherichia coli and Salmonella typhimurium), as well as against a pathogenic yeast (Candida albicans). All tested essential oils exhibited interesting antibacterial and antifungal activities against the assayed microorganisms.     

Biosynthesis of the irregular monoterpene artemisia ketone, the sesquiterpene germacrene D and other isoprenoids in Tanacetum vulgare L. (Asteraceae)

The incorporation of [1-13C]-labeled glucose into the irregular monoterpene artemisia ketone, the regular monoterpenes camphor and beta-thujone, the sesquiterpene germacrene D, the diterpene trans-phytol and beta-sitosterol and isofucosterol has been studied in axenic cultures of Tanacetum vulgare L. (Asteraceae). Quantitative 13C NMR spectroscopic analysis of the resulting labeling patterns showed that the isoprene units of the monoterpenes and the diterpene are formed via the methylerythritol phosphate (MEP) pathway, whereas the isoprene building blocks of the sesquiterpene and the sterols originate from the mevalonic acid (MVA) pathway.     

A study of variability in the MHC class II β1 and class I α2 domain exons of Atlantic salmon, Salmo salar L.

Variability in the most extracellular exons of Atlantic salmon MHC-Sasa class I and class II was studied by polymerase chain reaction (PCR) amplification followed by sequencing. The domains studied were class I α2 and class II β1. The material used was genomic DNA of fish, mainly derived from the major Norwegian breeding pool, supplemented with some material from a minor breeder and a local river strain. The analysis revealed extensive variation, most individuals being heterozygous with at least two variants.     

Unusual neuromuscular junctions in the heart of the crayfish (Procambarus clarkii)

Summary The neuromuscular junctions in the crayfish heart were studied with the electron microscope and were classified into two types based on the characteristics of the post-synaptic side. Type I junction was characterized by a mazy post-synaptic apparatus which has been referred to in this work as the junctional envelope, consisting of the cytoplasmic processes and/or lamellae of the muscle cell. Type II junction on the other hand, lacked the junctional envelope. The nerve terminals in both Type I and Type II junctions contained two types of synaptic vesicles: large granular and small agranular vesicles, which were about 1000 Å and 450 Å in diameter respectively. The physiological significance of these neuromuscular junctions and the nature of their synaptic vesicles are discussed.Acknowledgement. The author wishes to express sincere gratitude to Prof. T.Yamamoto for the kind encouragement and guidance during the course of this study.The presence of this unusual neuromuscular junction, coupled with the histological characteristics of heart muscles themselves (Komuro, 1968), may be involved in the different physiological properties of the crustacean heart. This subject will be discussed in a later publication by the author.     

Chemical Diversity of Essential Oils from Asteriscus graveolens (Forssk.) Less.: Identification of cis‐8‐Acetoxychrysanthenyl Acetate as a New Natural Component

Asteriscus graveolens is an endemic medicinal plant mainly distributed in south‐western Algeria and south‐eastern Morocco. The essential oils of leaves, stems, and flowers of A. graveolens had been studied by GC, GC/MS, and ¹³C‐NMR. The spectral data of two nerolidol derivatives, 6‐oxo‐ and 6‐hydroxycyclonerolidol, were reassigned by 1D‐ and 2D‐NMR spectroscopy. These compounds can be considered as chemical markers of this genus. The structure of a monoterpenic diester with a chrysanthenane skeleton, i.e., cis‐8‐acetoxychrysanthenyl acetate, was determined for the first time on the basis of GC/MS, and 1D‐ and 2D‐NMR. The stem and leaf oils were characterized by high content of oxygenated sesquiterpenes with 6‐oxo‐ and 6‐hydroxycyclonerolidol as major components, and the flower essential oils were dominated by the new monoterpenic compound cis‐8‐acetoxychrysanthenyl acetate.