Pantoea ananatis, a plant growth stimulating bacterium, and its metabolites isolated from Hydrocotyle umbellata (dollarweed).

A bacterium growing on infected leaves of Hydrocotyle umbellata, commonly known as dollarweed, was isolated and identified as Pantoea ananatis. An ethyl acetate extract of tryptic soy broth (TSB) liquid culture filtrate of the bacterium was subjected to silica gel chromatography to isolate bioactive molecules. Indole was isolated as the major compound that gave a distinct, foul odor to the extract, together with phenethyl alcohol, phenol, tryptophol, N-acyl-homoserine lactone, 3-(methylthio)-1-propanol, cyclo(L-pro-L-tyr), and cyclo(dehydroAla-L-Leu). This is the first report of the isolation of cyclo(dehydroAla-L-Leu) from a Pantoea species. Even though tryptophol is an intermediate in the indoleacetic acid (IAA) pathway, we were unable to detect or isolate IAA. We investigated the effect of P. ananatis inoculum on the growth of plants. Treatment of Lemna paucicostata Hegelm plants with 4 × 109 colony forming units of P. ananatis stimulated their growth by ca. five-fold after 13 days. After 13 days of treatment, some control plants were browning, but treated plants were greener and no plants were browning. The growth of both Cucumis sativus (cucumber) and Sorghum bicolor (sorghum) plants was increased by ca. 20 to 40%, depending on the growth parameter and species, when the rhizosphere was treated with the bacterium after germination at the same concentration. Plant growth promotion by Pantoea ananatis could be due to the provision of the IAA precursor indole.

Applying an internal transcribed spacer as a single molecular marker to differentiate between Tetraselmis and Chlorella species.

In the realm of applied phycology, algal physiology, and biochemistry publications, the absence of proper identification and documentation of microalgae is a common concern. This poses a significant challenge for non-specialists who struggle to identify numerous eukaryotic microalgae. However, a promising solution lies in employing an appropriate DNA barcoding technique and establishing comprehensive databases of reference sequences. To address this issue, we conducted a study focusing on the molecular characterization and strain identification of Tetraselmis and Chlorella species, utilizing the internal transcribed spacer (ITS) barcode approach. By analyzing the full nuclear ITS region through the Sanger sequencing approach, we obtained ITS barcodes that were subsequently compared with other ITS sequences of various Tetraselmis and Chlorella species. To ensure the reliability of our identification procedure, we conducted a meticulous comparison of the DNA alignment, constructed a phylogenetic tree, and determined the percentage of identical nucleotides. The findings of our study reveal the significant value of the ITS genomic region as a tool for distinguishing and identifying morphologically similar chlorophyta. Moreover, our results demonstrate that both the ITS1 and ITS2 regions are capable of effectively discriminating isolates from one another; however, ITS2 is preferred due to its greater intraspecific variation. These results underscore the indispensability of employing ITS barcoding in microalgae identification, highlighting the limitations of relying solely on morphological characterization.

Deoxyribonucleic Acid Barcoding for the Identification of Botanicals.

The Natural Herbal Products industry uses botanicals or herbs as raw materials for production of herbal products or dietary supplements. Recently, the demand for natural herbal products has increased tremendously and this has led to adulteration and to counterfeit herbal products. The present chapter deals with currently used molecular methods from "simple" single genomic regions to high-throughput whole genome or transcriptome sequencing methods used in the identification of botanicals.

Is Baikiain in Tara Flour a Causative Agent for the Adverse Events Associated with the Recalled Frozen French Lentil & Leek Crumbles Food Product? - A Working Hypothesis.

The French Lentil & Leek Crumbles frozen food product was recently recalled due to reports of gastrointestinal issues. So far, 393 adverse illness complaints and 133 hospitalizations have been reported from consumption of this food, and the tara (Tara spinosa) protein flour ingredient is hypothesized to be responsible. A multipronged approach resulted in identification of (S)-(-)-baikiain in tara as a compound of interest due to its abundance, possible metabolic fate, and close resemblance to irreversible inhibitors of L-pipecolate oxidase. Oral administration of baikiain in ND4 mice showed a statistically significant increase in blood ALT levels and a reduction in liver GSH.

A Comprehensive Workflow for the Analysis of Bio-Macromolecular Supplements: Case Study of 20 Whey Protein Products.

The presence of bio-macromolecules as major ingredients is a primary factor in marketing many biologically derived macromolecular supplements. Workflows for analyzing these supplements for quality assurance, adulteration, and other supply-chain difficulties must include a qualitative assessment of small-molecule and macromolecular components; however, no such integrated protocol has been reported for these bio-macromolecular supplements. Twenty whey protein supplements were analyzed using an integrated workflow to identify protein content, protein adulteration, inorganic elemental content, and macromolecular and small-molecule profiles. Orthogonal analytical methods were employed, including NMR profiling, LC-DAD-QToF analysis of small-molecule components, ICP-MS analysis of inorganic elements, determination of total protein content by a Bradford assay, SDS-PAGE protein profiling, and bottom-up shotgun proteomic analysis using LC-MS-MS. All 20 supplements showed a reduced protein content compared to the claimed content but no evidence of adulteration with protein from an unclaimed source. Many supplements included unlabeled small-molecule additives (but nontoxic) and significant deviations in metal content, highlighting the importance of both macromolecular and small-molecule analysis in the comprehensive profiling of macromolecular supplements. An orthogonal, integrated workflow allowed the detection of crucial product characteristics that would have remained unidentified using traditional workflows involving either analysis of small-molecule nutritional supplements or protein analysis.

The Low Copy Nuclear Gene Region, Granule Bound Starch Synthase (GBSS1), as a Novel Mini-DNA Barcode for the Identification of Different Sage (Salvia) Species.

Morphological similarity within species makes the identification and authentication of Salvia species challenging, especially in dietary supplements that contain processed root or leaf powder of different sage species. In the present study, the species discriminatory power of 2 potential DNA barcode regions from the nuclear genome was evaluated in 7 medicinally important Salvia species from the family Lamiaceae. The nuclear internal transcribed spacer 2 and the exon 9 - 14 region of low copy nuclear gene WAXY coding for granule-bound starch synthase 1 were tested for their species discrimination ability using distance, phylogenetic, and BLAST-based methods. A novel 2-step PCR method with 2 different annealing temperatures was developed to achieve maximum amplification from genomic DNA. The granule-bound starch synthase 1 region showed higher amplification and sequencing success rates, higher interspecific distances, and a perfect barcode gap for the tested species compared to the nuclear internal transcribed spacer 2. Hence, these novel mini-barcodes generated from low copy nuclear gene regions (granule-bound starch synthase) that were proven to be effective barcodes for identifying 7 Salvia species have potential for identification and authentication of other Salvia species.

Antimalarials and Phytotoxins from Botryosphaeria dothidea Identified from a Seed of Diseased Torreya taxifolia.

The metabolic pathways in the apicoplast organelle of Plasmodium parasites are similar to those in plastids in plant cells and are suitable targets for malaria drug discovery. Some phytotoxins released by plant pathogenic fungi have been known to target metabolic pathways of the plastid; thus, they may also serve as potential antimalarial drug leads. An EtOAc extract of the broth of the endophyte Botryosphaeria dothidea isolated from a seed collected from a Torreya taxifolia plant with disease symptoms, showed in vitro antimalarial and phytotoxic activities. Bioactivity-guided fractionation of the extract afforded a mixture of two known isomeric phytotoxins, FRT-A and flavipucine (or their enantiomers, sapinopyridione and (-)-flavipucine), and two new unstable γ-lactam alkaloids dothilactaenes A and B. The isomeric mixture of phytotoxins displayed strong phytotoxicity against both a dicot and a monocot and moderate cytotoxicity against a panel of cell lines. Dothilactaene A showed no activity. Dothilactaene B was isolated from the active fraction, which showed moderate in vitro antiplasmodial activity with high selectivity index. In spite of this activity, its instability and various other biological activities shown by related compounds would preclude it from being a viable antimalarial lead.

Antiplasmodial and Cytotoxic Cytochalasins from an Endophytic Fungus, Nemania sp. UM10M, Isolated from a Diseased Torreya taxifolia Leaf.

Bioassay-guided fractionation of an EtOAc extract of the broth of the endophytic fungus Nemania sp. UM10M (Xylariaceae) isolated from a diseased Torreya taxifolia leaf afforded three known cytochalasins, 19,20-epoxycytochalasins C (1) and D (2), and 18-deoxy-19,20-epoxy-cytochalasin C (3). All three compounds showed potent in vitro antiplasmodial activity and phytotoxicity with no cytotoxicity to Vero cells. These compounds exhibited moderate to weak cytotoxicity to some of the cell lines of a panel of solid tumor (SK-MEL, KB, BT-549, and SK-OV-3) and kidney epithelial cells (LLC-PK11). Evaluation of in vivo antimalarial activity of 19,20-epoxycytochalasin C (1) in a mouse model at 100 mg/kg dose showed that this compound had weak suppressive antiplasmodial activity and was toxic to animals.

Identification of Species in the Aromatic Spice Family Apiaceae Using DNA Mini-barcodes.

The species of the aromatic plant family Apiaceae are mainly used as spices and foods, but the family also includes medicinal and some poisonous plant species. Due to the similar chemical compounds or aroma and morphology, the poisonous species are often mistaken for the edible aromatic species. It is therefore imperative to correctly identify the species present at the initial raw stage samples to ensure product safety and efficacy. At the molecular level, plant species can be identified using DNA loci either from nuclear or plastid genome with easily available universal oligonucleotides, a technique called DNA barcoding. However, this is possible when single-species plant material is present but may not work on a mixture of plants species. Another disadvantage is that using universal oligonucleotides is of limited help, especially if the adulterating material is present in low quantities. On the other hand, if using the species-specific oligonucleotides, only single specific adulterating plant material could be detected and, consequently, the unexpected adulterants may go undetected. Therefore, in the current work, four degenerated oligonucleotides from ITS1 and ITS2 regions of the nuclear genome were designed that can bind to a variety of Apiaceae genera only and not to other genera belonging to different plant families. These family-specific oligonucleotides were able to amplify a diagnostic PCR product from 16 Apiaceae species that, upon sequencing, revealed the identity of the plant it was derived from. The size of these products is around 140 bp for ITS1 and approximately 80 bp for the ITS2 region. The size range of the amplified products falls in the category of a desired mini-barcode size to be used for damaged/fragmented DNA and next generation sequencing.

Essential Oils of Five Baccharis Species: Investigations on the Chemical Composition and Biological Activities.

This paper provides a comparative account of the essential oil chemical composition and biological activities of five Brazilian species of Baccharis (Asteraceae), namely B. microdonta, B. pauciflosculosa, B. punctulata, B. reticularioides, and B. sphenophylla. The chemical compositions of three species (B. pauciflosculosa, B. reticularioides, and B. sphenophylla) are reported for the first time. Analyses by GC/MS showed notable differences in the essential oil compositions of the five species. α-Pinene was observed in the highest concentration (24.50%) in B. reticularioides. Other major compounds included α-bisabolol (23.63%) in B. punctulata, spathulenol (24.74%) and kongol (22.22%) in B. microdonta, ß-pinene (18.33%) and limonene (18.77%) in B. pauciflosculosa, and ß-pinene (15.24%), limonene (14.33%), and spathulenol (13.15%) in B. sphenophylla. In vitro analyses for antimalarial, antitrypanosomal, and insecticidal activities were conducted for all of the species. B. microdonta and B. reticularioides showed good antitrypanosomal activities; B. sphenophylla showed insecticidal activities in fumigation bioassay against bed bugs; and B. pauciflosculosa, B. reticularioides, and B. sphenophylla exhibited moderate antimalarial activities. B. microdonta and B. punctulata showed cytotoxicity. The leaves and stems of all five species showed glandular trichomes and ducts as secretory structures. DNA barcoding successfully determined the main DNA sequences of the investigated species and enabled authenticating them.

Isolation of a phytotoxic isocoumarin from Diaporthe eres-infected Hedera helix (English ivy) and synthesis of its phytotoxic analogs.

BACKGROUND: The fungus Diaporthe eres was isolated from a fungal pathogen-infected leaf of Hedera helix (English ivy) exhibiting necrosis. It is hypothesized that the causative fungus produces phytotoxins as evidenced by necrotic lesions on the leaves. RESULTS: The fungus was isolated and grown in Czapek Dox broth culture medium and potato dextrose broth culture medium and identified as Diaporthe eres. The ethyl acetate extracts of the culture broths were phytotoxic to lettuce (Lactuca sativa) and bentgrass (Agrostis stolonifera). 3,4-Dihydro-8-hydroxy-3,5-dimethylisocoumarin (1) and tyrosol (2) were isolated and identified as the phytotoxic constituents. Six analogs of 3,4-dihydro-isocoumarin were synthesized and shown to be phytotoxic. The synthesized 3,4-dihydro-8-hydroxy-3,7-dimethylisocoumarin and 3,4-dihydro-8-hydroxy-3,3,7-trimethylisocoumarin were two- to three-fold more phytotoxic than the naturally occurring 1 in a Lemna paucicostata growth bioassay. CONCLUSION: Synthesis and herbicidal activities of the several new analogs of 1 are reported for the first time. These promising molecules should be used as templates for synthesis and testing of more analogs. © 2017 Society of Chemical Industry.

A single molecular marker to distinguish between species of Dioscorea.

Yams are species of the genus Dioscorea (family Dioscoreaceae), which consists of approximately 630 species. The majority of the world production of yams occurs in Africa with 58.8 million t annually, but they are also produced in the Americas and Asia. The saponins in yams have been reported to possess various properties to improve health. The tuber and aerial parts of various species often share morphological similarities, which can cause problems in the proper identification of sample material. For example, the rootstocks and aerial parts of Dioscorea villosa L. share similarities with Dioscorea polystachia Turcz. Dioscorea bulbifera L. may be mistaken for Dioscorea alata L. owing to similar morphologies. Various molecular analyses have been published to help with the identification of species and varieties within the genus Dioscorea. The multi-loci or single-locus analysis has resulted in varying success, some with only a limited discrimination rate. In the present study, a single nuclear genomic region, biparentally inherited, was analyzed for its usefulness as a molecular marker for species identification and discrimination between D. bulbifera, D. villosa, D. nipponica, D. alata, D. caucasica, and D. deltoidea samples. The results of this study show that the LFY genomic region can be useful as a molecular marker to distinguish between samples.

DNA Barcoding for the Identification of Botanicals in Herbal Medicine and Dietary Supplements: Strengths and Limitations.

In the past decades, the use of traditional medicine has increased globally, leading to a booming herbal medicine and dietary supplement industry. The increased popularity of herbal products has led to a rise in demand for botanical raw materials. Accurate identification of medicinal herbs is a legal requirement in most countries and prerequisite for delivering a quality product that meets consumer expectations. Traditional identification methods include botanical taxonomy, macroscopic and microscopic examination, and chemical methods. Advances in the identification of biological species using DNA-based techniques have led to the development of a DNA marker-based platform for authentication of plant materials. DNA barcoding, in particular, has been proposed as a means to identify herbal ingredients and to detect adulteration. However, general barcoding techniques using universal primers have been shown to provide mixed results with regard to data accuracy. Further technological advances such as mini-barcodes, digital polymerase chain reaction, and next generation sequencing provide additional tools for the authentication of herbs, and may be successful in identifying processed ingredients used in finished herbal products. This review gives an overview on the strengths and limitations of DNA barcoding techniques for botanical ingredient identification. Based on the available information, we do not recommend the use of universal primers for DNA barcoding of processed plant material as a sole means of species identification, but suggest an approach combining DNA-based methods using genus- or species-specific primers, chemical analysis, and microscopic and macroscopic methods for the successful authentication of botanical ingredients used in the herbal dietary supplement industry.

Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and α-bisabolene synthases.

Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including bioactive flavonoids, diterpene lactones, terpenoids and polysaccharides which accumulate in foliar tissues. Despite this chemical diversity, relatively few enzymes associated with any biosynthetic pathway from ginkgo have been characterized to date. In the present work, predicted transcripts potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoid compounds, including putative terpene synthases, were first identified by mining publicly-available G. biloba RNA-seq data sets. Recombinant enzyme studies with two of the TPS-like sequences led to the identification of GbTPS1 and GbTPS2, encoding farnesol and bisabolene synthases, respectively. Additionally, the phylogenetic analysis revealed the two terpene synthase genes as primitive genes that might have evolved from an ancestral diterpene synthase.

Phomalactone from a Phytopathogenic Fungus Infecting ZINNIA elegans (ASTERACEAE) Leaves.

Zinnia elegans Jacq. plants are infected by a fungus that causes dark red spots with necrosis on leaves, particularly in late spring to the middle of summer in the Mid-South of the United States. This fungal disease causes the leaves to wilt and eventually kills the plant. The fungus was isolated, cultured in potato dextrose broth, and identified as Nigrospora sphaerica by molecular techniques. Two major lactone metabolites (phomalactone and catenioblin A) were isolated from liquid culture of N. sphaerica isolated from Z. elegans. When injected into leaves of Z. elegans, phomalactone caused lesions similar to those of the fungus. The lesion sizes were proportional to the concentration of the phomalactone. Phomalactone, but not catenioblin A, was phytotoxic to Z. elegans and other plant species by inhibition of seedling growth and by causing electrolyte leakage from photosynthetic tissues of both Z. elegans leaves and cucumber cotyledons. This latter effect may be related to the wilting caused by the fungus in mature Z. elegans plants. Phomalactone was moderately fungicidal to Coletotrichum fragariae and two Phomopsis species, indicating that the compound may keep certain other fungi from encroaching into plant tissue that N. sphaerica has infected. Production of large amounts of phomalactone by N. sphaerica contributes to the pathogenic behavior of this fungus, and may have other ecological functions in the interaction of N. sphaerica with other fungi. This is the first report of isolation of catenioblin A from a plant pathogenic fungus. The function of catenioblin A is unclear, as it was neither significantly phyto- nor fungitoxic.

Molecular and phytochemical investigation of Angelica dahurica and Angelica pubescentis essential oils and their biological activity against Aedes aegypti, Stephanitis pyrioides, and Colletotrichum species.

In this study, Angelica dahurica and Angelica pubescentis root essential oils were investigated as pest management perspectives, and root samples were also analyzed genetically using the nuclear ribosomal internal transcribed spacer (ITS) region as a DNA barcode marker. A. pubescentis root essential oil demonstrated weak antifungal activity against Colletotrichum acutatum, Colletotrichum fragariae, and Colletotrichum gloeosporioides, whereas A. dahurica root essential oil did not show antifungal activity. Conversely, A. dahurica root essential oil demonstrated better biting deterrent and insecticidal activity against yellow fever mosquito, Aedes aegypti, and azalea lace bugs, Stephanitis pyrioides, than A. pubescentis root oil. The major compounds in the A. dahurica oil were found as α-pinene (46.3%), sabinene (9.3%), myrcene (5.5%), 1-dodecanol (5.2%), and terpinen-4-ol (4.9%). α-Pinene (37.6%), p-cymene (11.6%), limonene (8.7%), and cryptone (6.7%) were the major compounds found in the A. pubescentis oil. In mosquito bioassays, 1-dodecanol and 1-tridecanol showed antibiting deterrent activity similar to the positive control DEET (N,N-diethyl-3-methylbenzamide) at 25 nmol/cm(2) against Ae. aegypti, whereas only 1-tridecanol showed repellent activity in human-based cloth patch bioassay with minimum effective dosages (MED) of 0.086 ± 0.089 mg/cm(2) (DEET = 0.007 ± 0.003 mg/cm(2)). In larval bioassays, 1-tridecanol was more toxic with an LC50 value of 2.1 ppm than 1-dodecanol having an LC50 value of 5.2 ppm against 1-day-old Ae. aegypti larvae. 1-Dodecanol and 1-tridecanol could be useful for the natural mosquito control agents.

DNA barcoding of medicinal plant material for identification.

Because of the increasing demand for herbal remedies and for authentication of the source material, it is vital to provide a single database containing information about authentic plant materials and their potential adulterants. The database should provide DNA barcodes for data retrieval and similarity search. In order to obtain such barcodes, several molecular methods have been applied to develop markers that aid with the authentication and identification of medicinal plant materials. In this review, we discuss the genomic regions and molecular methods selected to provide barcodes, available databases and the potential future of barcoding using next generation sequencing.

Antifungal activity of extracts from endophytic fungi associated with Smallanthus maintained in vitro as autotrophic cultures and as pot plants in the greenhouse.

The endophytic fungal assemblages associated with Smallanthus sonchifolius (Poepp.) H. Rob. and Smallanthus uvedalius (L.) Mack. ex Small growing in vitro autotrophic cultures and in the greenhouse were identified and evaluated for their ability to produce bioactive compounds. A total of 25 isolates were recovered that were genetically closely related to species of the genera Bionectria , Cladosporium , Colletotrichum , Fusarium , Gibberella , Hypocrea , Lecythophora , Nigrospora , Plectosphaerella , and Trichoderma . The endophytic assemblages of S. sonchifolius presented a greater diversity than the group isolated from S. uvedalius and demonstrated the presence of dominant generalist fungi. Extracts of all fungi were screened against the fungal plant pathogens. Ten extracts (41.6%) displayed antifungal activities; some of them had a broad antifungal activity. The phylotypes Lecythophora sp. 1, Lecythophora sp. 2, and Fusarium oxysporum were isolated from in vitro autotrophic cultures and displayed antifungal activity. The presence of bioactive endophytic fungi within S. sonchifolius and S. uvedalius suggests an ecological advantage against pathogenic attacks. This study revealed reduced numbers of endophytes in association with both Smallanthus species in controlled cultivation conditions compared with the endophytic communities of hosts collected in the wild environments. Even as reduced endophytic communities, these fungi continue to provide chemical protection for the host.

Molecular analysis of genetic fidelity in Cannabis sativa L. plants grown from synthetic (encapsulated) seeds following in vitro storage.

The increasing utilization of synthetic (encapsulated) seeds for germplasm conservation and propagation necessitates the assessment of genetic stability of conserved propagules following their plantlet conversion. We have assessed the genetic stability of synthetic seeds of Cannabis sativa L. during in vitro multiplication and storage for 6 months at different growth conditions using inter simple sequence repeat (ISSR) DNA fingerprinting. Molecular analysis of randomly selected plants from each batch was conducted using 14 ISSR markers. Of the 14 primers tested, nine produced 40 distinct and reproducible bands. All the ISSR profiles from in vitro stored plants were monomorphic and comparable to the mother plant which confirms the genetic stability among the clones. GC analysis of six major cannabinoids [Δ(9)-tetrahydrocannabinol, tetrahydrocannabivarin, cannabidiol, cannabichromene, cannabigerol and cannabinol] showed homogeneity in the re-grown clones and the mother plant with insignificant differences in cannabinoids content, thereby confirming the stability of plants derived from synthetic seeds following 6 months storage.

Molecular analyses of the Chinese herb Leigongteng (Tripterygium wilfordii Hook.f.).

Tripterygium wilfordii Hook.f., known as Leigongteng (Thunder God Vine) in traditional Chinese medicine, has attracted much attention for its applications in relieving autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus, and for treating cancer. Molecular analyses of the ITS and 5S rDNA sequences indicate that T. hypoglaucum and T. doianum are not distinct from T. wilfordii, while T. regelii should be recognized as a separate species. The results also demonstrate potential value of rDNA sequence data in forensic detection of adulterants derived from Celastrus angulatus in commercial samples of Leigongteng.