Essential‐Oil Composition of Daucus carota ssp. major (Pastinocello Carrot) and Nine Different Commercial Varieties of Daucus carota ssp. sativus Fruits

The chemical composition of the essential oils obtained by hydrodistillation from the pastinocello carrot, Daucus carota ssp. major (Vis.) Arcang . (flowers and achenes), and from nine different commercial varieties of D. carota L. ssp. sativus (achenes) was investigated by GC/MS analyses. Selective breeding over centuries of a naturally occurring subspecies of the wild carrot, D. carota L. ssp. sativus, has produced the common garden vegetable with reduced bitterness, increased sweetness, and minimized woody core. On the other hand, the cultivation of the pastinocello carrot has been abandoned, even if, recently, there has been renewed interest in the development of this species, which risks genetic erosion. The cultivated carrot (D. carota ssp. sativus) and the pastinocello carrot (D. carota ssp. major) were classified as different subspecies of the same species. This close relationship between the two subspecies urged us to compare the chemical composition of their essential oils, to evaluate the differences. The main essential‐oil constituents isolated from the pastinocello fruits were geranyl acetate (34.2%), α‐pinene (12.9%), geraniol (6.9%), myrcene (4.7%), epi‐α‐bisabolol (4.5%), sabinene (3.3%), and limonene (3.0%). The fruit essential oils of the nine commercial varieties of D. carota ssp. sativus were very different from that of pastinocello, as also confirmed by multivariate statistical analyses.     

Une nouvelle sous-espèce de loup (Canis lupus maximus nov. subsp.) dans le Pléistocène supérieur d’Europe occidentale

The evolutionary history of the extant wolf (Canis lupus sensu largo) and its ancestors is characterised by changes in body size. A chronocline has been established based on these changes, but its temporal resolution is poorly defined. New morphometric analyses conducted on Late Pleistocene remains from southern France have permitted the diagnosis of a new subspecies of wolf, Canis lupus maximus nov. subsp. This new subspecies is statistically larger than all other known fossil and extant wolves from Western Europe. The diagnosis is based on biometric data collected on wolf remains from Jaurens cave (Nespouls, Corrèze, France). The paleoenvironmental context of the biogeographic range of this subspecies is discussed.     

Inhibitory Activity and Chemical Characterization of Daucus carota subsp. maximus Essential Oils

The essential oils (EOs) of green seeds from Daucus carota subsp. maximus growing wild in Pantelleria Island (Sicily, Italy) were characterized. EOs were extracted by steam distillation, examined for their inhibitory properties against food‐borne Gram‐positive and Gram‐negative bacteria and analyzed for the chemical composition by gas chromatography (GC) and mass spectrometry (MS). Undiluted EOs showed a large inhibition spectrum against Gram‐positive strains and also vs. Acinetobacter spp. and Stenotrophomonas maltophilia. The minimum inhibition concentration (MIC) was in the range 1.25 – 2.50 μl/ml for the most sensitive strains. The chemical analysis indicated that D. carota subsp. maximus EOs included 34 compounds (five monoterpene hydrocarbons, six oxygenated monoterpenes, 14 sesquiterpene hydrocarbons, four oxygenated sesquiterpenes, camphorene and four other compounds), accounting for 95.48% of the total oil, and that the major chemicals were carotol, β‐bisabolene, and isoelemicin.     

Zur systematischen Stellung vonValeriana pancicii Halácsy &Baldacci undV. bertiscea Pančić

Summary Morphological and ecological studies as well as investigations of essential oils reveal thatValeriana pancicii Hal. &Bald., an endemic of southern Montenegro, must be considered a subspecies ofValeriana celtica. ThusV. celtica includes 3 subspecies: subsp.celtica (subsp.pennina Vierh.) endemic in the western, subsp.norica in the eastern Alpes and subsp.pancicii in Montenegro (Abb. 2). While the two alpine subspecies are polyploids, subsp.pancicii proved to be diploid on x=12, and may be supposed to stand closest to the ancestor of theV. celtica-complex. The occurence of subsp.pancicii in Montenegro seems to indicate an East Mediterranean-montane descent of the complex.A comparison ofValeriana montana L. andV. bertiscea Pan., described from Montenegro on the basis of herbarium specimens, and a study of populations on the locus classicus hardly justify taxonomic separation fromV. montana.     

Comparative chemical composition of Agastache mexicana subsp. mexicana and A. mexicana subsp. xolocotziana

A comparative chemical analysis of Agastache mexicana subsp. mexicana and A. mexicana subsp. xolocotziana reveals that their methanol extracts constituents were very similar, with acacetin and (2-acetyl)-7-O-glucosyl acacetin being the most abundant compounds obtained. These results are consistent with the information reported for other species of Agastache. However, GS-MS analyses showed that methyl chavicol, limonene and linalool were the main constituents of the essential oils of A. mexicana subsp. mexicana, while pulegone, menthone and isopulegone were the major constituents found in A. mexicana subsp. xolocotziana. Furthermore, a different composition was found in their respective hexane extracts. These chemical composition dissimilarities between the two taxa support their recognition as distinct subspecies.     

Chemical and genetic diversity of two Mediterranean subspecies of Teucrium polium L.

Chemical and genetic diversity of Teucrium polium L. subsp. polium from western Algeria and T. polium L. subsp. capitatum from Corsica were investigated. Diversity within and among the two populations of subspecies was assessed according to the chemical composition of their essential oils and the genetic diversity. Chemical analysis was performed using a combination of capillary GC-RI and GC/MS after fractionation using column chromatography. Genetic structures were mapped using three polymorphic genetic markers: two chloroplast markers (RPL32-TRNL and TRNL-F) and ribosomal nuclear markers (ITS region). The statistical analysis showed that both subspecies were clearly distinguished by these chemical and genetic markers. The oil chemical compositions differed qualitatively and quantitatively between the subspecies. Both collective oils were dominated by hydrocarbon compounds however the Algerian sample oils exhibited higher amounts of hydrocarbon sesquiterpenes than those of Corsica (31.2 g/100 g vs. 4.4 g/100 g) while the latter displayed higher amounts of hydrocarbon monoterpenes than the first (59.3 g/100 g vs. 34.3 g/100 g). Neighbor-joining, Maximum likelihood and Bayesian trees constructed from chloroplast markers and nuclear ITS region sequences showed the existence of two groups associated with taxonomic and chemical characteristics. The study indicated that variation in the essential oil composition within subspecies depends on genetic background. The samples of subsp. capitatum from Corsica are a homogeneous group, in contrast to samples of subsp. polium from Algeria which were clustered in two groups. Chemical and genetic diversity of Algerian populations could be explained by geographical isolation of the populations. In addition, the morphological polymorphism observed throughout the colour of flowers could be explained by environmental parameters as well as the soil pH.     

Chemical variability in volatile composition between several Italian accessions of Siler montanum (S. montanum subsp. montanum and S. montanum subsp. siculum)

Laserpitium siler, currently recognized as Siler montanum, is a polymorphic species belonging to the Apiaceae family and distributed in mountains of Southern Europe. In the present work we have analysed five accessions from Italy (Piemonte, Valle d'Aosta and Abruzzo) belonging to S. montanum subsp. montanum and subsp. siculum for the essential oil composition with the aim to find correlations between chemical data and taxonomic relationships. Results, obtained by gas chromatography-mass spectrometry (GC-MS), showed a significant variability, with the subsp. siculum characterized by one chemotype (sabinene/perilla aldehyde/chamazulene), and the subsp. montanum belonging to three different chemotypes (I (E)-anethole, II sabinene, III limonene). Principal component analysis (PCA) was used to identify the chemical differences among the five accessions according to geographical origin and subspecies.     

Mitochondrial genome diversity among cultivars of daucus carota (ssp. sativus) and their wild relatives

Summary Restriction fragment patterns of mitochondrial DNAs (mtDNAs) from 13 carrot cultivars (Daucus carota ssp. sativus), wild carrot (ssp. carota), ssp. gummifer, and D. capillifolius were compared with each other using four restriction endonucleases. The mtDNAs of the 13 carrot cultivars could be classified into three distinct types — I, II and III — and were also clearly distinguishable from the mtDNAs of wild carrot (type IV), gummifer (V) and D. capillifolius (VI). The proportions of common restriction fragments (F values) shared by two of the three mtDNA types (I, II and III) of carrot cultivars were approximately 0.5–0.6. The F values were 0.4–0.5 for mitochondrial genomes between wild carrot, ssp. gummifer and D. capillifolius. The mitochondrial genomes between wild carrot and the carrot cultivars showed closer homologies those between wild carrot, ssp. gummifer, and D. capillifolius. The diversity of the mitochondrial genomes among the carrot cultivars is too high to presume that it was generated from the cytoplasm of only one common ancestor during the relatively short history of carrot breeding. These results suggested that the three types of cytoplasms found in the carrot cultivars might have existed in a prototype of D. carota in pre-historical times.     

Electrophoretic study of isozyme patterns in some wild populations of Aubrieta columnae Guss. (Cruciferae)

Aubrieta columnae Guss. is currently divided into three subspecies: A. columnae subsp. columnae, A. columnae subsp. italica, both found as isolated and fragmented populations in rocky habitats of Central and Southern Apennines (Italy), and A. columnae subsp. croatica found in the Balkan region. In order to gain information about the degree of genetic variability and to clarify taxonomic relationships among these taxa, we studied the isozyme patterns at 8 marker loci of 376 individuals from 8 populations by means of starch gel electrophoresis. Data analysis by using Wright's F-statistics and UPGMA clustering method was performed. The results show: 1) a general deviation from Hardy–Weinberg equilibrium within each subspecies; 2) a lesser genetic variation in populations occurring in habitats characterized by milder climatic conditions and relatively small seasonal variations; 3) a relatively high degree of differentiation between the three subspecies; 4) the possible common transadriatic origin of A. columnae subsp. italica and A. columnae subsp. croatica; 5) the possible origin of A. columnae subsp. columnae from A. columnae subsp. italica; and 6) that the current taxonomic status of A. columnae may be substantially confirmed, even if the findings are from a limited number of loci explored.     

Ribosomal protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for phylogenety-based subspecies resolution of Bifidobacterium longum

The taxonomic positions of the subspecies of Bifidobacterium longum (B. longum subsp. longum, subsp. infantis, and subsp. suis) have been controversial. A current proposal is that the former two species “B. infantis” and “B. suis” be unified with B. longum and all three reclassified as three subspecies. To test this proposal, ribosomal protein profiling as observed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied to the classification of 17 strains of B. longum, including three subspecies. Among 41 different kinds of ribosomal proteins selected as biomarkers whose masses were calculated from their amino acid sequences, 31-41 ribosomal proteins were observed in sample strains with the same masses as the references. The high matching rate indicates high conservation of ribosomal proteins within the sample strains, and therefore strongly supports the unification of the former species. However, the masses of some ribosomal proteins varied within species. The phylogenetic tree constructed from the profiles of ribosomal proteins matched the references, showing a clear cluster of the subsp. longum and the subsp. infantis strains. This result supports the proposal to reclassify B. longum into subsp. longum and subsp. infantis. The subsp. suis strains formed an individual sub-cluster within the infantis cluster. However, their ribosomal proteins have both characters of longum and infantis types. This result suggests that the taxonomic position of the subsp. suis should be reconsidered.     

VARIATION OF PLASTID AND MITOCHONDRIAL DNAS IN THE GENUS DAUCUS

Plastid and mitochondrial DNAs from suspension cultures of Daucus carota subsp. sativus cv. Danvers, D. carota subsp. gummifer, D. capillifolius, and D. pusillus were compared by restriction endonuclease fragment analysis. Organelles isolated from protoplasts of suspension cultures were purified using a one-step sucrose gradient. Plastid DNA fragment patterns for subsp. sativus, subsp. gummifer, and capillifolius were indistinguishable in Pstl, SalI and XhoI enzyme digests. No variation was detected in BamHI or HindIII digests between subsp. sativus and capillifolius. Pusillus plastid DNA varied significantly when compared to the other Daucus cultures. The size of the plastid genome of each species as determined by fragment addition, was estimated at 155 kb. Restriction digests of the mitochondrial DNAs generated a large number of fragments which when totaled established a size of 386–468 kb for the genomes. Densitometer scans of the fragment patterns indicate the bands were present in variable stoichiometry. Up to 26% of the fragments generated by PstI, SalI and HindIII digests were homologous in size among the four mitochondrial genomes. A further comparison of mitochondrial fragments indicated a closer homology of subsp. sativus to capillifolius than to subsp. gummifer or pusillus as was also found with the plastid genomes.     

Parallel Evolution Under Domestication and Phenotypic Differentiation of the Cultivated Subspecies of <Emphasis Type="Italic">Cucurbita pepo</Emphasis> (Cucurbitaceae)

Parallel Evolution under Domestication and Phenotypic Differentiation of the Cultivated Subspecies of Cucurbita pepo (Cucurbitaceae). Cucurbita pepo (pumpkin, squash, gourd, Cucurbitaceae) is an ancient North American domesticate of considerable economic importance. Based on molecular genetic polymorphisms, two cultivated lineages of this species, each consisting of very many edible–fruited cultigens, have been recognized, C. pepo subsp. pepo and C. pepo subsp. texana. However, the phenotypic commonalities and differences between these two subspecies have not as yet been systematically collected and organized. Among the evolutionary developments common to the two subspecies are the increased size of the plant parts, less plant branching, and premature loss of chlorophyll in the exocarp of the fruits. In both subspecies, bush growth habit, conferred by allele Bu, is common to the cultigens grown for consumption of the immature fruits, as is the deviation from the 1:1 ratio of fruit length to fruit width. A major characteristic differentiating between the edible–fruited cultigens of the respective subspecies are the longitudinal protrusions, in subsp. pepo, versus depressions, in subsp. texana, of the fruit surface corresponding with the subsurface primary carpellary vein tracts. Subsp. pepo also has larger fruits and larger and longer seeds. In addition, some alleles affecting stem color, leaf mottling, multiple flower bud production, and fruit characteristics are frequently occurring to nearly fixed in one subspecies but are rare to less common in the other.     

Larvicidal activity of Amyris balsamifera,Daucus carota and Pogostemon cablin essential oils and their components against Culex pipiens pallens

Larvicidal activities of Amyris balsamifera, Daucus carota, and Pogostemon cablin essential oils were tested against Culex pipiens pallens. All three oils showed 100% larvicidal activity against C. pipiens pallens at 0.1 mg/mL. Among the tested oils, the larvicidal activity of D. carota oil was the strongest followed by P. cablin and A. balsamifera. Four active compounds such as β-eudesmol, elemol, patchoulol, and carotol were isolated from the three oils by open column chromatography. These compounds showed > 90% mortality against C. pipiens pallens at 0.1 mg/mL. In acute toxicity testing of the water flea, Daphnia magna, P. cablin oil was the most toxic followed by A. balsamifera, and D. carota. Among the isolated compounds, carotol was the most toxic to water fleas. The residues of P. cablin, A. balsamifera, and D. carota in water were 67.8%, 59.5%, and 51.2% at 2 days after treatment, respectively. High concentrations of elemol and patchoulol were detected 2 days after treatment compared to those of β-eudesmol and elemol. Whole oils and compounds tested were detected at < 50% after 7 days in water.     

Phylogenetic Analysis and Polyphasic Characterization of Clavibacter michiganensis Strains Isolated from Tomato Seeds Reveal that Nonpathogenic Strains Are Distinct from C. michiganensis subsp. michiganensis

The genus Clavibacter comprises one species and five subspecies of plant-pathogenic bacteria, four of which are classified as quarantine organisms due to the high economic threat they pose. Clavibacter michiganensis subsp. michiganensis is one of the most important pathogens of tomato, but the recommended diagnostic tools are not satisfactory due to false-negative and/or -positive results. To provide a robust analysis of the genetic relatedness among a worldwide collection of C. michiganensis subsp. michiganensis strains, relatives (strains from the four other C. michiganensis subspecies), and nonpathogenic Clavibacter-like strains isolated from tomato, we performed multilocus sequence-based analysis and typing (MLSA and MLST) based on six housekeeping genes (atpD, dnaK, gyrB, ppK, recA, and rpoB). We compared this “framework” with phenotypic and genotypic characteristics such as pathogenicity on tomato, reaction to two antisera by immunofluorescence and to five PCR identification tests, and the presence of four genes encoding the main C. michiganensis subsp. michiganensis pathogenicity determinants. We showed that C. michiganensis subsp. michiganensis is monophyletic and is distinct from its closest taxonomic neighbors. The nonpathogenic Clavibacter-like strains were identified as C. michiganensis using 16S rRNA gene sequencing. These strains, while cross-reacting with C. michiganensis subsp. michiganensis identification tools, are phylogenetically distinct from the pathogenic strains but belong to the C. michiganensis clade. C. michiganensis subsp. michiganensis clonal complexes linked strains from highly diverse geographical origins and also strains isolated over long periods of time in the same location. This illustrates the importance of seed transmission in the worldwide dispersion of this pathogen and its survival and adaptation abilities in a new environment once introduced.     

Genetic relationships and evolution in <Emphasis Type="Italic">Cucurbita pepo</Emphasis> (pumpkin,squash, gourd) as revealed by simple sequence repeat polymorphisms

Genetic relationships among 104 accessions of Cucurbita pepo were assessed from polymorphisms in 134 SSR (microsatellite) and four SCAR loci, yielding a total of 418 alleles, distributed among all 20 linkage groups. Genetic distance values were calculated, a dendrogram constructed, and principal coordinate analyses conducted. The results showed 100 of the accessions as distributed among three clusters representing each of the recognized subspecies, pepo, texana, and fraterna. The remaining four accessions, all having very small, round, striped fruits, assumed central positions between the two cultivated subspecies, pepo and texana, suggesting that they are relicts of undescribed wild ancestors of the two domesticated subspecies. In both, subsp. texana and subsp. pepo, accessions belonging to the same cultivar-group (fruit shape) associated with one another. Within subsp. pepo, accessions grown for their seeds or that are generalists, used for both seed and fruit consumption, assumed central positions. Specialized accessions, grown exclusively for consumption of their young fruits, or their mature fruit flesh, or seed oil extraction, tended to assume outlying positions, and the different specializations radiated outward from the center in different directions. Accessions of the longest-fruited cultivar-group, Cocozelle, radiated bidirectionally, indicating independent selection events for long fruits in subsp. pepo probably driven by a common desire to consume the young fruits. Among the accessions tested, there was no evidence for crossing between subspecies after domestication.     

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.     

A proposal to unify two subspecies of Staphylococcus equorum: Staphylococcus equorum subsp. equorum and Staphylococcus equorum subsp. linens

Twelve isolates from jeotgal, a Korean high-salt-fermented seafood, identified as Staphylococcus equorum were compared by phenotypic and genotypic methods to determine their precise taxonomic identities at the subspecies level. Four strains and three strains had complete 16S rRNA gene sequence matches with S. equorum subsp. equorum DSM 20674^T and S. equorum subsp. linens DSM 15097^T, respectively. Five strains showed 99.9 % identity with the sequences of both type strains. In our DNA–DNA hybridization analyses among two type strains and two isolates, the similarities were over 72 % and were higher than the similarities presented at the subspecies proposal. Physiological characteristics such as sugar utilization, β-galactosidase activity, novobiocin resistance and salt tolerance, which were adopted for subspecies separation, could not be applied to assign the isolates to a taxonomic unit. Antibiotic susceptibility, hemolytic activity, biofilm formation and protein profiles did not present markers to divide the isolates into either of the subspecies. Multilocus sequence typing of the sequences of the 16S rRNA gene and five housekeeping genes did not produce any coherent relationship among the isolates and type strains. Repetitive element-PCR fingerprinting using ERIC (enterobacterial repetitive intergenic consensus) primers classified 12 isolates to three genotypes, and the genotypes of both type strains coincided with two isolates expressing different characteristics. Based on these phenotypic and genotypic analyses results, we propose to unify the present two subspecies of S. equorum into one species, S. equorum.     

Comparison of Salmonella enterica Serovar Typhimurium LT2 and Non-LT2 Salmonella Genomic Sequences, and Genotyping of Salmonellae by Using PCR

Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.