Assessment of the DNA barcoding for identification of Trigonostigma somphongsi,a critically endangered species in Thailand

Trigonostigma somphongsi, a critically endangered species, is a rare and endemic fish in Thailand. This species had disappeared from its natural habitat for 20 years until 2006. The DNA barcodes or the fragments of cytochrome c oxidase I (COI) of T. somphongsi were investigated for species identification. The remaining two native species in the genus Trigonostigma, T. heteromorpha and T. espei were also identified using Boraras urophthalmoides as an outgroup species. The 707-bp fragments were successfully amplified and sequenced in all fifteen fish samples. In the genus Trigonostigma, the genetic distance within and between species ranged from 0.000 to 0.005 and 0.016 to 0.039, respectively. The lowest genetic distance (0.016) was between T. heteromorpha and T. espei, while the highest genetic distance (0.039) was between T. somphongsi and T. espei, followed by T. somphongsi and T. heteromorpha (0.035). The phylogenetic analysis showed that the relationship between the three Trigonostigma species (T. somphongsi was clearly separated from T. heteromorpha and T. espei) agreed with the morphological characteristics. These results suggest that DNA barcoding is an effective approach to identify Trigonostigma species for use in the conservation and management of fisheries.     

Molecular phylogeny of Phoma and allied anamorph genera: Towards a reclassification of the Phoma complex

The present generic concept of Phoma is broadly defined, with nine sections being recognised based on morphological characters. Teleomorph states of Phoma have been described in the genera Didymella, Leptosphaeria, Pleospora and Mycosphaerella, indicating that Phoma anamorphs represent a polyphyletic group. In an attempt to delineate generic boundaries, representative strains of the various Phoma sections and allied coelomycetous genera were included for study. Sequence data of the 18S nrDNA (SSU) and the 28S nrDNA (LSU) regions of 18 Phoma strains included were compared with those of representative strains of 39 allied anamorph genera, including Ascochyta, Coniothyrium, Deuterophoma, Microsphaeropsis, Pleurophoma, Pyrenochaeta, and 11 teleomorph genera. The type species of the Phoma sections Phoma, Phyllostictoides, Sclerophomella, Macrospora and Peyronellaea grouped in a subclade in the Pleosporales with the type species of Ascochyta and Microsphaeropsis. The new family Didymellaceae is proposed to accommodate these Phoma sections and related anamorph genera. The present study demonstrated that Phoma radicina, the type species of Phoma sect. Paraphoma and Phoma heteromorphospora, the type species of Phoma sect. Heterospora can be assigned to the Phaeosphaeriaceae and Leptosphaeriaceae respectively.     

Peroxidase: a marker for Ascochyta blight resistance in chickpea

On the basis of incidence of appearance of Ascochyta blight symptoms after artificial inoculation of 25-day-old chickpea seedlings with 10 different pathotypes of Ascochyta rabiei, GL94011, PBG5 and C214 have been classified as resistant, moderately resistant and susceptible, respectively, to Ascochyta blight. In none of the pathotypes, peroxidase (PO) activity could be detected in culture medium and mycelium. Healthy tissues of GL94011 have almost three times the PO activity in comparison with that of C214. Resistant and moderately resistant genotypes showed 30–60% upregulation of PO activity against infection by A. rabiei whereas it was only 3–6% in susceptible genotype C214. These results indicate the possibility of using PO as a marker of Ascochyta blight resistance.     

Activation of superoxide production and differential exocytosis in polymorphonuclear leukocytes by cytochalasins A,B, C,D and E: Effects of various ions

All of the common cytochalasins activate superoxide anion release and exocytosis of β-N-acetylglucosaminidase and lysozyme from guinea-pig polymorphonuclear leukocytes (neutrophils) incubated in a buffered sucrose medium. Half-maximal activation of both processes is produced by approx. 2 μM cytochalasin A, C >μM cytochalasin B ? 4–5 μM cytochalasin D, E. While maximal rates of O₂^? release and extents of exocytosis require extracellular calcium (1–2 mM), replacing sucrose with monovalent cation chlorides is inhibitory to neutrophil activation by cytochalasins. Na⁺, K⁺ or choline inhibited either cytochalasin B- or E-stimulated O₂^? production with IC₅₀ values of 5–10 mM and inhibition occurs whether Cl^?, NO₃^? or SCN^? is the anion added with Na⁺ or K⁺. Release of β-N-acetylglucosaminidase in control or cytochalasin B-stimulated cells is inhibited by NaCl (IC₅₀ ≈ 10 mM), while cytochalasin E-stimulated exocytosis is reduced less and K⁺ or choline chloride are ineffective in inhibiting either cytochalasin B- or E-stimulated exocytosis. Release of β-glucuronidase, myeloperoxidase or acid phosphatase from neutrophils incubated in buffered sucrose is not stimulated by cytochalasin B. Stimulation of either O₂^? or β-N-acetylglucosaminidase release by low concentrations of cytochalasin A is followed by inhibition of each at higher concentrations. It appears that all cytochalasins can activate both NAD(P)H oxidase and selective degranulation of neutrophils incubated in salt-restricted media and that differential inhibition of these two processes by monovalent cations and/or anions is produced at some step(s) subsequent to cytochalasin interaction with the cell.     

Studies on the Host Range of the Hyperparasite Aphanocladium album

The uredospores of the 14 rust species tested and an Ascochyta, sp. were heavily parasitized by A. album. The teliospores of 3 Ustilaginales species were slightly parasitized. Some other important plant parasites were inhibited at distance, some others did not show any damage.     

Killing potential protist predators as a survival strategy of the newly described dinoflagellate Alexandrium pohangense

Blooms caused by some species belonging to the dinoflagellate genus Alexandrium are known to cause large-scale mortality of fish. Thus, the dynamics of these species is important and of concern to scientists, officials, and people in the aquaculture industry. To understand the dynamics of such species, their growth and mortality due to predation need to be assessed. The newly described dinoflagellate Alexandrium pohangense is known to grow slowly, with a maximum autotrophic growth rate of 0.1 d⁻¹. Thus, it may not form bloom patches if its mortality due to predation is high. Therefore, to explore the mortality of A. pohangense due to predation, feeding on this species by the common heterotrophic dinoflagellates Gyrodinium dominans, Gyrodinium moestrupii, Luciella masanensis, Noctiluca scintillans, Oxyrrhis marina, Oblea rotunda, Polykrikos kofoidii, and Pfiesteria piscicida, as well as by the ciliate Tiarina fusus, was examined. None of these potential predators was able to feed on A. pohangense. In contrast, these potential predators were killed and their bodies were dissolved when incubated with A. pohangense cells or cell-free culture filtrates. The survival of G. moestrupii, O. marina, P. kofoidii, and T. fusus on incubation with 10 cells ml⁻¹ of A. pohangense was 20–60%, while that at the equivalent culture filtrates was 20–70%. With increasing A. pohangense cell-concentration (up to 1000 cells ml⁻¹ or equivalent culture filtrates), the survival rate of G. moestrupii, O. marina, P. kofoidii, and T. fusus rapidly decreased. The lethal concentration (LC₅₀) for G. moestrupii, O. marina, P. kofoidii, and T. fusus at the elapsed time of 24 h with A. pohangense cells (cultures of 11.4, 13.3, 1.6, and 3.3 cells ml⁻¹, respectively) was lower than that with A. pohangense filtrates (culture filtrates of 35.5, 30.6, 5.5, and 5.0 cells ml⁻¹, respectively). Furthermore, most of the ciliates and heterotrophic dinoflagellates in the water collected from the coast of Tongyoung, Korea, were killed when incubated with cultures of 1000 A. pohangense cells ml⁻¹ and equivalent culture filtrates. The relatively slow growing A. pohangense may form blooms by reducing mortality due to predation through killing potential protist predators.     

Analysis of Indole-3-Acetic Acid and Related Indoles in Culture Medium from Azospirillum lipoferum and Azospirillum brasilense

Analysis of neutral and acidic ethyl acetate extracts from culture medium of Azospirillum brasilense 703Ebc by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol, indole-3-methanol, and indole-3-lactic acid. IAA in media of 20 strains of A. brasilense and Azospirillum lipoferum was analyzed quantitatively by both the colorimetric Salkowski assay and HPLC-based isotopic dilution procedures. There was little correlation between the estimates obtained with the two procedures. For instance, the Salkowski assay suggested that the culture medium from A. brasilense 703Ebc contained 26.1 μg of IAA ml⁻¹, whereas HPLC revealed the presence of only 0.5 μg of IAA ml⁻¹. Equivalent estimates with A. brasilense 204Ed were 10.5 and 0.01 μg of IAA ml⁻¹, respectively. The data demonstrate that the Salkowski assay is not a reliable method for measuring the IAA content of Azospirillum culture medium and that estimates in excess of 10 μg of IAA ml⁻¹ should be viewed with particular caution. Metabolism of [2′-¹⁴C]IAA by A. brasilense 703Ebc yielded radiolabeled indole-3-methanol, whereas roots of maize (Zea mays L.) seedlings gave rise to [¹⁴C]oxindole-3-acetic acid and an array of polar metabolites. Metabolism of [2′-¹⁴C]IAA by maize roots inoculated with A. brasilense 703Ebc produced a metabolic profile characteristic of maize rather than Azospirillum species.     

Cytochalasin binding in lymphocytes and polymorphonuclear leukocytes

The binding of [³H]cytochalasin B (CB) to intact cells was compared in lymphocytes, polymorphonuclear leukocytes (PMNLs) and erythrocytes over a broad range of cytochalasin concentrations. Binding curves consistent with the presence of high and low affinity binding sites were demonstrated in all three cell types. However, in contrast to observations in erythrocytes, in lymphocytes and PMNLs CB binding was unaffected by d-glucose. p-Hydroxymercuribenzoate and p-hydroxymercurisulfonate were only partially inhibitory and unlabeled cytochalasins E, D and A (CE, CD, CA) inhibited [³H]CB binding more effectively than unlabeled CB. While attempts to demonstrate that plasma membrane-rich subcellular fractions from lymphocytes selectively bind [³H]CB were inconclusive, radioautographic studies on unbroken cells indicated that most or all of the high affinity CB-binding sites in lymphocytes and PMNLs were in close proximity to the cell surface.     

A Real-Time PCR-Based Semi-Quantitative Breakpoint to Aid in Molecular Identification of Urinary Tract Infections

This study presents a novel approach to aid in diagnosis of urinary tract infections (UTIs). A real-time PCR assay was used to screen for culture-positive urinary specimens and to identify the causative uropathogen. Semi-quantitative breakpoints were used to screen for significant bacteriuria (presence of ≥10⁵ CFU/ml of uropathogens) or low-level bacteriuria (containing between 10³ and 10⁴ CFU/ml of uropathogens). The 16S rDNA-based assay could identify the most prevalent uropathogens using probes for Escherichia coli, Pseudomonas species, Pseudomonas aeruginosa, Staphylococcus species, Staphylococcus aureus, Enterococcus species and Streptococcus species. 330 urinary specimens were analysed and results were compared with conventional urine culture. Using a PCR Ct value of 25 as semi-quantitative breakpoint for significant bacteriuria resulted in a sensitivity and specificity of 97% and 80%, respectively. In 78% of the samples with monomicrobial infections the assay contained probes to detect the bacteria present in the urine specimens and 99% of these uropathogens was correctly identified. Concluding, this proof-of-concept approach demonstrates that the assay can distinguish bacteriuria from no bacteriuria as well as detect the involved uropathogen within 4 hours after sampling, allowing adequate therapy decisions within the same day as well as drastically reduce consequent urine culturing.     

Aspergillus flavus colonization and aflatoxin B1 formation in barley grain during interaction with other fungi

Colonization of barley grain by Aspergillus flavus and formation of aflatoxin B₁ in the presence of Penicillium verrucosum, Fusarium sporotrichioides, and Hyphopichia burtonii were studied over a three-week period in all combinations of 20 or 30 °C and 0.97, 0.95 or 0.90 a_w. Grain colonization was assessed initially by observing hyphal extension on the grain surface, using scanning electron microscopy, and then from the proportion of seeds infected and numbers of colony forming units (cfu) formed. Aflatoxin b¹ concentrations were determined by enzyme linked immunosorbent assay using a monoclonal antibody. These studies showed that interaction between A. flavus and other fungi in paired culture had different effects on both colonization and aflatoxin formation depending on the species involved and environmental conditions. Germination of A. flavus spores was unaffected by the presence of other species on the grain surface. Subsequently, three principal patterns of A. flavus colonization of barley grain were observed through the incubation period in the presence of other fungal species: (a) colonization unaffected by the presence of other species; (b) colonization initially slower in the presence of other species but later differing little from pure cultures; and (c) colonization adversely affected by the presence of other species. Five main patterns of aflatoxin B₁ production were observed relative to pure culture but with no consistent relationship with species, a_w, temperature or incubation period; (a) little changed; (b) increased slowly; (c) decreased; (d) enhanced; and (e, f) increased initially but later decreased to (e) the same level as in pure culture or (f) to less than in pure culture. Generally, production of aflatoxin B₁ by A. flavus was less than in pure culture but sometimes was changed only slightly by the presence of P. verrucosum, F. sporotrichioides or H. burtonii or was temporarily enhanced.     

Effect of triarimol on sterol and fatty acid composition of three species of Chlorella

The concentration of triarimol giving ca 50% inhibition of growth was different for each of 3 species of Chlorella [C. emersonii, 1 mg/l. (1.5 × 10^?6 M), C. ellipsoidea 10 mg/l. (3 × 10^?5 M), C. sorokiniana, 2 mg/l. (6 × 10^?6 M)]. The total lipid of 3 species of Chlorella grown in a culture medium containing triarimol were analysed for chlorophyll, fatty acids and sterol composition. Growth rates were studied in the presence of different concentrations of triarimol. The growth rates of the 3 species were differentially inhibited by triarimol. The growth of Chlorella sorokiniana was 50% inhibited by 2 mg/l. triarimol but 20 mg/l. did not produce a cessation of growth. The greatest inhibition of growth rates and chlorophyll content was observed in Chlorella emersonii. The quantity of unsaturated fatty acids was increased by triarimol treatment in all 3 species of Chlorella. Triarimol strongly inhibited 14α-demethylation in Chlorella emersonii, and C. ellipsoidea and less in C. sorokiniana, resulting in accumulation of 14α-methyl sterols. Triarimol also inhibited the second alkylation of the side chain in C. ellipsoidea and C. emersonii. The introduction of the 22-double bond was inhibited in all 3 species of Chlorella studied. Although some differences were apparent, the effect of triarimol was quite similar to that of triparanol and AY-9944 in these 3 species of Chlorella.     

Evaluation of Rpf protein of Micrococcus luteus for cultivation of soil actinobacteria

Rpf protein, a kind of resuscitation promoting factor, was first found in the culture supernatant of Micrococcus luteus. It can resuscitate the growth of M. luteus in “viable but non-culture, VBNC” state and promote the growth of Gram-positive bacteria with high G + C content. This paper investigates the resuscitating activity of M. luteus ACCC 41016^T Rpf protein, which was heterologously expressed in E. coli, to cells of M. luteus ACCC 41016^T and Rhodococcus marinonascens HBUM200062 in VBNC state, and examines the effect on the cultivation of actinobacteria in soil. The results showed that the recombinant Rpf protein had resuscitation effect on M. luteus ACCC 41016^T and R. marinonascens HBUM200062 in VBNC state. 83 strains of actinobacteria, which were distributed in 9 families and 12 genera, were isolated from the experimental group with recombinant Rpf protein in the culture medium. A total of 41 strains of bacteria, which were distributed in 8 families and 9 genera, were isolated from the control group without Rpf protein. The experimental group showed richer species diversity than the control group. Two rare actinobacteria, namely HBUM206391^T and HBUM206404^T, were obtained in the experimental group supplemented with Rpf protein. Both may be potential new species of Actinomadura and Actinokineospora, indicating that the recombinant expression of M. luteus ACCC 41016^T Rpf protein can effectively promote the isolation and culture of actinobacteria in soil.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day^?1) and yield (60 μg chlorophyll/ml culture) than in pure cultures (0.4 day^?1 and 10 μg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Chemostats

The enhanced mineralization of organic nitrogen by bacteriophagous protozoa is thought to favor the nitrification process in soils, in which nitrifying bacteria have to compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the bacteriovorous flagellate Adriamonas peritocrescens on the competition for limiting amounts of ammonium between the ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis was studied in the presence of Nitrobacter winogradskyi in continuous cultures at dilution rates of 0.004 and 0.01 h^-1. The ammonium concentration in the reservoir was maintained at 2 mM, whereas the glucose concentration was increased stepwise from 0 to 7 mM. A. globiformis won the competition for limiting amounts of ammonium when the glucose concentration in the reservoirs increased, in agreement with previously described experiments in which the flagellates were not included. The numbers of nitrifying bacteria decreased as the numbers of heterotrophic bacteria rose with increasing glucose concentrations. Critical C/N ratios, i.e., ratios between glucose and ammonium in the reservoirs at which no nitrate was found in the culture vessels, of 12.5 and 10.5 were determined at dilution rates of 0.004 and 0.01 h^-1, respectively. Below these critical values, coexistence of the competing species was found. The numbers of nitrifying bacteria decreased more in the presence of flagellates than in their absence, presumably by selective predation on the nitrifying bacteria, either in the liquid culture or on the glass wall of the culture vessels. Despite this, the rate of nitrate production did not decrease more in the presence of flagellates than in their absence. This demonstrates that no correlation has to be expected between numbers of nitrifying bacteria and their activity and that a constant nitrification rate per cell cannot be assumed for nitrifying bacteria. Above the critical C/N ratios, low numbers of nitrifying bacteria were still found in the culture vessels, probably because of attachment of the nitrifying bacteria to the glass wall of the culture vessels. Like the numbers of heterotrophic bacteria, the numbers of flagellates increased when the glucose concentrations in the reservoirs increased. Numbers of 2 × 10⁵ and 12 × 10⁵ flagellates ml^-1 were found at 7 mM glucose at dilution rates of 0.004 and 0.01 h^-1, respectively. It was concluded that the critical C/N ratios were practically unaffected by the presence of protozoa. Although nitrate production rates were equal in the presence and absence of flagellates, the numbers of nitrifying bacteria decreased more strongly in their presence. This indicates a higher activity per nitrifying cell in the presence of flagellates.     

Ascochitine Production by Fungi Responsible for Ascochyta Diseases of Pea

Ascochitine production by 24 isolates of three fungal species causing the Ascochyta disease complex in pea plants, was examined. All eight isolates of Ascochyta pisi Lib. secreted ascochitine to liquid nutrient medium in amounts of 0.1 to 1.2 mg/l. However, no ascochitine was found in culture filtrates of Mycosphaerella pinodes (Berk. and Blox.) Vestergr. or of Phoma medicaginis var. pinodella (Jon.) Boerema although the aggressiveness of these two species as pathogens was shown to be higher than that of A. pisi. Individual isolates of each of the three fungal species examined did not differ in their pathogenicity.     

Phylogeny and taxonomy of Halimeda incrassata,including descriptions of H. kanaloana and H. heteromorpha spp. nov. (Bryopsidales,Chlorophyta)

The tropical green algal genus Halimeda is one of the best studied examples of pseudo-cryptic diversity within the algae. Previous molecular and morphometric studies revealed that within Halimeda section Rhipsalis, Halimeda incrassata included three pseudo-cryptic entities and that the morphological boundaries between H. incrassata and Halimeda melanesica were ill-defined. In this paper, the taxonomy of H. incrassata is revised: two pseudo-cryptic entities are described as new species, Halimeda kanaloana and Halimed heteromorpha, while H. incrassata is redefined to encompass a single, monophyletic entity. Similarities and differences between the three species and H. melanesica are discussed. Monophyly of H. heteromorpha, which was questioned in a former study, is reinvestigated using sets of 32 ITS1–ITS2 and 21 plastid rps3 sequences and various alignment and inference methods. The phylogenetic relationships within Halimeda section Rhipsalis are inferred from nuclear 18S–ITS1–5.8S–ITS2 and concatenated plastid sequences (tufA & rpl5–rps8–infA) and interpreted in a biogeographic context.     

Production of cytochalasins C,D & E from dematiaceous hyphomycetes

One hundred isolates of 27 species belonging to 13 genera of dematiaceous hyphomycetes were screened for production of cytochalasins C, D and E. Only two isolates ofBipolaris neergaardii and one isolate ofPhoma herbarum produced cytochalasins C and D. Also, cytochalasin E was produced by two isolates ofAlternaria chlamydospora, and one isolate ofCochliobolus tuberculatus. This is the first report about the production of cytochalasins C, D and E by these species of dematiaceous hyphomycetes.     

Effect of cytochalasins on surfactant release from alveolar type II cells

Cytochalasins enhanced surfactant secretion from primary cultures of [³H]choline-labeled type II epithelial cells from the rat. Cytochalasins A, B, C, D and dihydrocytochalasin B enhanced secretion of phosphatidyl-[³H]choline ([³H]PC) in a dose-dependent manner with EC₅₀ values of 1, 2, 0.5, 0.1 and 1 μM for cytochalasins A, B, C, D and dihydrocytochalasin B, respectively. Only cytochalasin A caused significant cytotoxicity as determined by release of the intracellular enzyme lactate dehydrogenase (EC 1.1.1.17). Dose responses of surfactant release induced by cytochalasins B, C and D were biphasic; maximal release was observed between 0.1–1.0 μM for cytochalasins C and D between 1 and 10 μM for cytochalasin B. Secretion decreased toward control levels at concentrations of cytochalasin above these maximal concentrations. Increased rates of [³H]PC release were noted between 1 and 3 h after exposure to cytochalasin D. Increased rates of surfactant release induced by cytochalasin D were additive to release induced by the β-adrenergic agonist, terbutaline, or forskolin, although cytochalasin D had no direct effect on cytosolic cyclic AMP levels. Changes in cell shape and microfilament organization were observed by phase-contrast microscopy and fluorescence microscopy using rhodamine-conjugated phalloidin after exposure of the isolated type II cells to cytochalasin D. Disruption of microfilaments associated with lamellar bodies of the purified type II cells occurred after treatment with cytochalasin D. Cytochalasin D augmented surfactant release from purified type II cells and disrupted the microfilament structure of those cells, supporting the hypothesis that alterations in microfilaments are associated with surfactant release.     

Identification of Carnobacterium Species by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR

The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNA^Ala and tRNA^Ile, which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.     

Endogenous isoflavone methylation correlates with the in vitro rooting phases of Spartium junceum L. (Leguminosae)

Spartium junceum L. (Leguminosae) is a perennial shrub, native to the Mediterranean region in southern Europe, widespread in all the Italian regions and, as a leguminous species, it has a high isoflavone content. An in vitro culture protocol was developed for this species starting from stem nodal sections of in vivo plants, and isoflavone components of the in vitro cultured tissues were studied by means of High Performance Liquid Chromatography (HPLC) analytical techniques. Two main isoflavones were detected in the S. junceum tissues during the in vitro propagation phases: Genistein (4′,5,7-Trihydroxyisoflavone), already reported in this species, and its methylated form 4′,5,7-Trimethoxyisoflavone, detected for the first time in this plant species (0.750 ± 0.02 mg g⁻¹ dry tissue). The presence of both of these compounds in S. junceum tissues was consistently detected during the in vitro multiplication phase. The absence of the methylated form within plant tissues in the early phases of the in vitro adventitious root formation was correlated with its negative effect displayed on root induction and initiation phases, while its presence in the final “root manifestation” phase influenced positively the rooting process. The unmethylated form, although detectable in tissues in the precocious rooting phases, was no longer present in the final rooting phase. Its effect on rooting, however, proved always to be beneficial.