Nuclear Ribosomal DNA ITS Sequences Research Articles

Antifungal Potential of Cladosporium sp. (Endophytic fungi) Associated with Olea europaea L. Leaves

In the leaves of Olea europaea L. Olive trees an endophytic fungus was discovered. Cladosporium sp. was identified to be the fungus based on its morphological characteristics and nuclear ribosomal DNA ITS sequence analysis and was registered in NCBI as the Cladosporium genus has been registered under the number (0P939922.1) The species was not specified, and it was considered of unknown species after comparing it to global isolates. In comparison to olive leaf extract, Cladosporium sp. including total flavonoid, total phenolic, total terpenoid, and total saponins, Which were 121.9%, 198.1%, 89.13%, and 29.87 % respectively compared to its content in olive leaf extract, which was 61.54 %, 67.88 % , 17.1%, and 20.19% respectively. The Cladosporium sp. extract inhibited the growth of 27 isolates belonging to different species of candida which were Candida albicans , C. lypolitica , C. tropicalis , C. sphaerica , C. krusei , C. guilliermondii , C. parapsilosis , C. norvegicus , C. glabrata , and C. kefyr , the inhibition effects increased with increasing concentration to reach the highest level to suppress fungal growth when concentrated 30 mg/ml. This proves the antifungal potential of endophytic fungi in the future.

Isolation and Characterization of Novel Huperzine-Producing Endophytic Fungi from Lycopodiaceae Species.

Huperzine A (HupA) is an important drug for treating Alzheimer's disease (AD) and is primarily extracted from the Huperzia serrata (Lycopodiaceae). Failures in the chemical synthesis of Hup and in vitro culture have put H. serrata in danger of extinction, and there is a need for an extensive investigation of Hup from alternative perspectives. The aim of this study is to identify endophytic fungi that produce high Hup or simultaneously produce many types of Hup and have high genetic stability derived from other Lycopodiaceae species as a source of materials for natural Hup production. In this work, Hup-producing endophytic fungi were isolated from three species: Lycopodium clavatum, Phlegmariurus squarrosus, and P. phlegmaria. Of these, L. clavatum and P. squarrosus were confirmed as novel sources of Hup-producing fungi. Based on morphological characteristics and nuclear ribosomal DNA ITS sequences, four endophytic fungi Colletotrichum siamense THG1-17, Epicoccum sorghinum THG01-18, Phoma sp. TKH3-2, and Phyllosticta sp. THG2-27 were firstly isolated from these Lycopodiaceae plants, which were capable of simultaneously producing both HupA and HupB, as evidenced by high-performance liquid chromatography analysis. The four strains showed stability in Hup yield over 50 generations of culture with an in vitro storage period of 3 months. These isolated fungi will provide a new source of materials for further research to develop drugs containing HupA as well as HupB for AD treatment in the future.

Plastome evolution in the genus Sium (Apiaceae, Oenantheae) inferred from phylogenomic and comparative analyses

BackgroundSium L. (Apiaceae) is a small genus distributed primarily in Eurasia, with one species also occurring in North America. Recently, its circumscription has been revised to include 10 species, however, the phylogenetic relationships within its two inclusive clades were poorly supported or collapsed in previous studies based on nuclear ribosomal DNA ITS or cpDNA sequences. To identify molecular markers suitable for future intraspecific phylogeographic and population genetic studies, and to evaluate the efficacy of plastome in resolving the phylogenetic relationships of the genus, the complete chloroplast (cp) genomes of six Sium species were sequenced.ResultsThe Sium plastomes exhibited typical quadripartite structures of Apiaceae and most other higher plant plastid DNAs, and were relatively conserved in their size (153,029–155,006 bp), gene arrangement and content (with 114 unique genes). A total of 61–67 SSRs, along with 12 highly divergent regions (trnQ, trnG-atpA, trnE-trnT, rps4-trnT, accD-psbI, rpl16, ycf1-ndhF, ndhF-rpl32, rpl32-trnL, ndhE-ndhG, ycf1a and ycf1b) were discovered in the plastomes. No significant IR length variation was detected showing that plastome evolution was conserved within this genus. Phylogenomic analysis based on whole chloroplast genome sequences produced a highly resolved phylogenetic tree, in which the monophyly of Sium, as well as the sister relationship of its two inclusive clades were strongly supported.ConclusionsThe plastome sequences could greatly improve phylogenetic resolution, and will provide genomic resources and potential markers useful for future studies of the genus.

Phylogenetic analysis of some fungi species in West Kazakhstan based on nuclear ribosomal DNA ITS sequences

In this study, phylogenetic analysis of some fungal species distributed in West Kazakhstan was performed based on ITS sequences. All of the mushroom samples were collected from different regions of West Kazakhstan and brought to the laboratory. Total genomic DNA was extracted using a GeneMark commercial kit. ITS1 and ITS4 primers were used for the amplification of the ITS region in PCR analyses. The resulting DNA sequences were then edited using BioEdit and FinchTV. For phylogenetic analysis used MEGA 6.0 program. As a result of the study, ITS sequences ranged from 532 to 715 nucleotides, while the divergence values of the sequences differed between 0.000 and 0.468. The maximum likelihood tree constructed using ITS sequences consists of two clades. According to the phylogenetic analysis results obtained using other fungal species, ITS results were found to be a good indicator for the differentiation of fungal genera.

Phylogenomic insight into dysploidy, speciation, and plastome evolution of a small Mediterranean genus Reichardia (Cichorieae; Asteraceae)

Reichardia Roth is a small Mediterranean genus comprising ten homogeneous species with basic chromosome numbers of 7, 8, and 9. To assess the plastid genome evolution and differentiation of Reichardia species, we assembled the complete plastome sequences of seven Reichardia and two Launaea species and conducted various phylogenomic analyses comparatively with nuclear ribosomal DNA ITS sequences. Reichardia and Launaea plastomes were highly conserved in gene content and order, containing 130 genes. Plastid phylogenomic reconstruction strongly suggested that Reichardia was a sister to Launaea, and its common ancestor initially diverged into two major lineages: the first containing species with n = 8 chromosomes exclusively, and the other with n = 9, 8, and 7 chromosomes. Although the ancestral Reichardia karyotype was suggested to most likely be n = 9 from ancestral chromosome number reconstruction, the pattern of descending dysploidy indicated by the phylogenetic trees based on nuclear ribosomal DNA ITS was less evident in the trees based on the plastome. Possible reasons for these findings are discussed.

A note on systematic position of Indian species of Cyathocline (Compositae)

The Genus Cyathocline has been merged with Blumea following molecular phylogenetic evidence (Plastid trnL-F and nuclear ribosomal DNA ITS sequences data). While the type species, C. purpurea has been transferred to Blumea, other species have not been transferred. In light of this, new combinations are made here for C. lutea & C. manilaliana. C. purpurea var. alba and C. purpurea var. bicolor are synonymized under Blumea purpurea. B. manilaliana is reported here the first time from Maharashtra state.

Five novel and highly efficient endophytic fungi isolated from Huperzia serrata expressing huperzine A for the treatment of Alzheimer’s disease

Huperzine A (Hup A) is an important drug for treating Alzheimer's disease (AD) and mainly extracted from the Huperzia serrata (Thunb.) Trevis. (Lycopodiaceae) (HS). Nevertheless, the content of Hup A in HS is very low of 0.007% with growing circle of 8 to 10 years, and the chemical synthesis of Hup A still has some insurmountable limitations in the industrialized production. So, the available resources of Hup A for clinical treatment of AD are scarce. The purpose of this work was to construct a biosynthesis platform based on the endophytic fungi from HS. In this work, five endophytic fungi Mucor racemosus NSH-D, Mucor fragilis NSY-1, Fusarium verticillioides NSH-5, Fusarium oxysporum NSG-1, and Trichoderma harzianum NSW-V were firstly found and isolated from the Chinese folk medicine HS, which were identified according to their morphological characteristics and nuclear ribosomal DNA ITS sequences. The highest efficient fungus could effectively biosynthesize Hup A in a liquid culture of 319.8 ± 0.17 mg/L which were 112 times higher than that of other reported conventional endophytic fungi. Moreover, these fungi with higher hereditary stability could possess the initial expressing ability of Hup A after 40 generations, and the expressed Hup A from these biosynthesis systems has prior physicochemical properties, a better inhibition activity of acetylcholinesterase and a lower cytotoxicity compared with the listed active pharmaceutical ingredients (APIs) of Hup A. These results provide promising alternative resources for producing Hup A at an industrial scale by biosynthesis, and it may also shed light on millions of AD patients. KEY POINTS: • Five novel endophytic fungi with high stability could highly express prior Hup A Graphical abstract.

Dissimilar molecular and morphological patterns in an introgressed peripheral population of a sand dune species (Armeria pungens, Plumbaginaceae).

Introgression is a poorly understood evolutionary outcome of hybridisation because it may remain largely undetected whenever it involves the transfer of small parts of the genome from one species to another. Aiming to understand the early stages of this process, a putative case from the southernmost border of the Armeria pungens range from its congener A.macrophylla is revisited following the discovery of a subpopulation that does not show phenotypic signs of introgression and resembles typical A.pungens. We analysed morphometrics, nuclear ribosomal DNA ITS and plastid DNA (trnL-trnF) sequences, genome size, 45S and 5S rDNA loci-FISH data and nrDNA IGS sequences. Within the study site, most individuals match morphologies of either of the two hybridising species, particularly the new subpopulation, with intermediate phenotypes being scarce. This pattern does not fully fit molecular evidence revealing two ITS ribotypes co-occurring intragenomically in most plants from the study site and one single plastid haplotype. Genome size and structural features of the IGS sequences both indicate that A.pungens from the study site is genetically more similar to its sympatric congener than to the remainder of its conspecifics. Introgression of A.macrophylla into A.pungens and plastid capture explain all the evidence analysed. However, important features to understand the origin and fate of the introgressed population, such as the degree and direction of introgression, which are important for understanding early stages of hybridisation in plants with low reproductive barriers, should be addressed with new data.

Paris variabilis (Melanthiaceae), a new species from southwestern China

Paris variabilis, a new species from the Wumengshan Mountains, southwestern China, is described and illustrated. The new species is placed in Paris section Euthyra. The new taxon was determined to be most morphologically similar to P. vietnamensis but differs in its oblong leaf blades with an acute apex, stamens 2–4 × petal number, greenish yellow filaments and an enlarged, purplish red style base. The phylogenetic placement of this species was assessed based on nuclear ribosomal ITS DNA sequences data. The results of morphological and phylogenetic analyses support the status of the taxon as a new species.

Biosynthesis of AgNPs in Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) (P-AgNPs) Using the Endophytic Fungus Fusarium solani Isolated from an Endangered Medicinal Plant Plumbago rosea and Their Anti Bacterial and Anticancer Activity on Human Breast Cancer Cells (MCF-7)

Abstract—Among the most hopeful biomaterials, metallic nanoparticles with antibacterial and anticancer properties are expected to open new avenues to fight and prevent various cancer and infectious diseases. The aim of the current study was the biosynthesis of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone-AgNPs (P-AgNPs) using the endophytic fungus Fusarium solani isolated from an endangered medicinal plant Plumbago rosea. The fungus was identified according to its morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. The synthesized P-AgNPs were initially noticed through visual color change from yellow to reddish brown and further confirmed by surface plasmonic resonance (SPR) band at 450 nm using UV-visible spectroscopy. Fourier transform infrared (FTIR) analysis showed amines and amides that are responsible for the stabilization of P-AgNPs. High-resolution scanning electron microscopy (HRSEM) showed that P-AgNPs were well dispersed, spherical, and well within the range of 23.2, 33.2 and 33.7 nm. X-ray diffraction (XRD) study revealed crystalline nature of P-AgNPs. These P-AgNPs displayed good antibacterial activity in dose dependant manner. The synthesized P-AgNPs exhibited dose-depended cytotoxicity against MCF-7 human breast cancer cells, the inhibitory IC50 were found to be 14.5 μg/mL. These nanoparticles were further studied for their anticancer activity and showed high selective toxicity against the MCF-7 breast cancer cell line. The anticancer activity also executed through inhibiting the cells at the phase DNA synthesis.

Aphyllophoroid fungi in insular woodlands of eastern Ukraine.

BackgroundFungi play crucial roles in ecosystems and are among the species-richest organism groups on Earth. However, knowledge on their occurrence lags behind the data for animals and plants. Recent analyses of fungal occurrence data from Western, Central and Northern Europe provided important insights into response of fungi to global warming. The consequences of the global changes for biodiversity on a larger geographical scale are not yet understood. Landscapes of Eastern Europe and particularly of eastern Ukraine, with their specific geological history, vegetation and climate, can add substantially new information about fungal diversity in Europe.New informationWe describe the dataset and provide a checklist of aphyllophoroid fungi (non-gilled macroscopic Basidiomycota) from eastern Ukraine sampled in 16 areas between 2007 and 2011. The dataset was managed on the PlutoF biodiversity workbench (http://dx.doi.org/10.15156/BIO/587471) and can also be accessed via Global Biodiversity Information Facility (GBIF, parts of datasets https://doi.org/10.15468/kuspj6 and https://doi.org/10.15468/h7qtfd). This dataset includes 3418 occurences, namely 2727 specimens and 691 observations of fructifications belonging to 349 species of fungi. With these data, the digitised CWU herbarium (V. N. Karazin Kharkiv National University, Ukraine) doubled in size A most detailed description of the substrate's properties and habitat for each record is provided. The specimen records are supplemented by 26 nuclear ribosomal DNA ITS sequences and six 28S sequences. Additionally, 287 photographs depicting diagnostic macro- and microscopic features of fungal fruitbodies as well as studied habitats are linked to the dataset. Most of the specimens have at least one mention in literature and relevant references are displayed as associated with specimen data. In total, 16 publication references are linked to the dataset. The dataset sheds new light on the fungal diversity of Eastern Europe. It is expected to complement other public sources of fungal occurrence information on continental and global levels in addressing macroecological and biogeographical questions.

The Namib-Thar Desert Disjunction in Dactyliandra (Cucurbitaceae) is the Result of a Recent Introduction to India

Abstract Depending on taxonomic concept, the genus Dactyliandra (Cucurbitaceae) comprises up to four species: two from Southwest Africa, D. luederitziana (Namibia) and D. welwitschii (Angola), and two very locally distributed and poorly known taxa from Northeast Africa, D. nigrescens and D. stefaninii. In addition to these African populations, D. welwitschii was also reported as a possibly native species from the Thar desert of Northwestern India and Pakistan ca. 8,000 km away from the Namib range. A phylogenetic analysis of plastid and nuclear ribosomal ITS DNA sequences including all Dactyliandra species and D. welwitschii from the Thar desert revealed that this striking disjunction is of very recent origin. Dactyliandra is monophyletic only after exclusion of the East African taxa, which are best placed in Trochomeria. The species Dactyliandra welwitschii is monophyletic and includes the Indian plants, which group with a sample from an Angolan population. We hypothesize that this unexpected connection ...

Floral similarity and vegetative divergence in a new species of Bletia (Orchidaceae) from Mexico

Bletia mixtecana, a new species from Oaxaca, Mexico is described and illustrated. This species is florally similar to B. parkinsonii but differs in its aerial thickened stems (‘pseudobulbs’) and several floral attributes. A phylogenetic analysis of nuclear ribosomal ITS DNA sequences indicates that B. mixtecana and B. parkinsonii are not closely related, suggesting that floral similarity represents either parallelism or shared ancestral (symplesiomorphic) traits. The new species is a strict gypsophile known only from two populations and it qualifies as endangered based on the small number of known populations and individuals, high habitat specificity and the observed loss of plants at one of the two known locations.

Phylogenetic relationship of some Onobrychis taxa naturally grown in Turkey based on morphology and nuclear ribosomal DNA ITS Sequences

Phylogenetic relationships were studied based on morphology and nuclear ribosomal DNA internal transcript spacer (nrDNA ITS) sequences in 49 populations of 36 Onobrychis taxa belonging to 5 sections (Dendobrychis, Lophobrychis, Onobrychis, Heliobrychis and Hymenobrychis) naturally grown in Turkey. The morphological data (46 quantitative characters) were standardized and submitted for PCA analysis before the performance of hierarchical clustering analysis. In addition, a 577-bp region of the nrDNA ITS sequences was compared to generate a dendrogram using the neighbor-joining method. According to the obtained results, the Onobrychis genus is clearly divided into the Onobrychis and Sisyrosema subgenera both molecularly and morphologically; however, in terms of molecular data, while the Onobrychis subgenus showed polyphyletic features, the Sisyrosema subgenus had monophyletic features. Additionally, long-wing taxa belonging to Dendobrychis and Onobrychis and O. crista-galli belonging to Lophobrychis located in the same clade in Onobrychis showed polyphyletic features. This finding was confirmed in terms of long-wing taxa as well as morphology and indicated that these three sections were entangled with each other under the Onobrychis subgenus.

Phylogeny and biogeography of Alyssum (Brassicaceae) based on nuclear ribosomal ITS DNA sequences.

The genus Alyssum consists of about 195 species native to Europe, Asia and northern Africa. All species were assigned to six sections. Previous molecular phylogeny studies indicate that Alyssum is polyphyletic. However, the divergence time and dispersal of the genus are not well studied. In this study, the phylogenetic relationships within the genus Alyssum were studied with nrDNA ITS sequences obtained from five sections. The divergence time was estimated by fossil calibration and the biogeography was examined by spread analysis. The phylogeny indicated two main lineages: lineage 1 includes the section of Alyssum, Gamosepalum and Psilonema; lineage 2 includes the section of Odontarrhena, Meniocus and Clypeola. The phylogenetic relationship was not congruent with the previous sectional classifications. The age of Alyssum was dated to the upper Miocene. Molecular data suggested the diversification of Alyssum in Mediterranean areas and wide-ranging distribution such as North Africa, eastward into Central Asia and immigration into North America. Climatic aridification and arid/semiarid areas established in the Pliocene/Pleistocene could have provided favourable conditions for the migration and diversification of Alyssum.

Ecological specialization in Trebouxia (Trebouxiophyceae) photobionts of Ramalina menziesii (Ramalinaceae) across six range-covering ecoregions of western North America.

• Premise of the study: Many lichens exhibit extensive ranges spanning several ecoregions. It has been hypothesized that this wide ecological amplitude is facilitated by fungal association with locally adapted photobiont strains.• Methods: We studied the identity and geographic distribution of photobionts of the widely distributed North American lichen Ramalina menziesii based on rbcL (chloroplast DNA) and nuclear ribosomal ITS DNA sequences. To test for ecological specialization, we associate photobiont genotypes with local climate and phorophyte.• Key results: Of the photobiont lineages of R. menziesii, 94% belong to a clade including Trebouxia decolorans. The remaining are related to T. jamesii. The photobionts showed (1) significant structure according to ecoregion and phorophyte species and (2) genetic associations with phorophyte species and climate.• Conclusions: Geography, climate, and ecological specialization shape genetic differentiation of lichen photobionts. One great advantage of independent dispersal of the fungus is symbiotic association with locally adapted photobiont strains.

Molecular phylogeny of Daucus (Apiaceae): Evidence from nuclear ribosomal DNA ITS sequences

The Apiaceae genus Daucus consists of approximately 25 species (including carrots) which are characterized by the presence of bracts in pedunculate umbels, dorsally compressed mericarps, hairs on primary ridges, and uniseriately arranged spines on the secondary ridges of the fruit. Taxonomically, Daucus has been considered to be one of the most problematic genera in the Apiaceae due to the highly variable fruit morphology. Despite taxonomic controversy and economic importance of the genus, no rigorously constructed estimate of phylogenetic relationships exists. To examine generic limit and relationships among species of Daucus and its putatively related taxa, phylogenetic analyses of characters derived from nuclear ribosomal DNA ITS sequences were conducted. Two major clades emerged within Daucus, but neither of them have been previously recognized using morphological characters. The phylogeny also provides taxonomic status of recently reported new species of Daucus, D. arcanus and D. conchitae.

An Endophytic Sanguinarine-Producing Fungus from Macleaya cordata, Fusarium proliferatum BLH51

Fermentation processes using sanguinarine-producing fungi other than Macleaya cordata may be an alternative way to produce sanguinarine (SA), which is a quaternary benzo[c]phenanthridine alkaloid possessing antibacterial, anthelmintic, and anti-inflammatory properties. In this study, a SA-producing endophytic fungus strain BLH51 was isolated from the leaves of M. cordata grown in the Dabie Mountain, China. Strain BLH51 produced SA when grown in potato dextrose liquid medium. The amount of SA produced by this endophytic fungus was quantified to be 178 μg/L by HPLC, substantially lower than that produced by the host tissue. The fungal SA--which was analyzed by thin layer chromatography and high-performance liquid chromatography--was shown to be identical to authentic SA. Strain BLH51 was identified as Fusarium proliferatum based on the morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. To the best of our knowledge, this is the first report concerning the isolation and identification of endophytic SA-producing fungi from the host plant, which further proved that endophytic fungi are valuable reservoirs of bioactive compounds.

Recircumscription of Begonia sect. Baryandra (Begoniaceae): evidence from molecular data

BackgroundBegonia sect. Diploclinium is a ‘dust-bin’ section for species retaining pleisiomorphic characters and lacking novel synapomorphic characters used to delimit other Asian sections in Begonia. Part of this large and polymorphous section is transferred to Begonia sect. Baryandra in a move towards a more natural classification for the genus.ResultsPhylogenetic analysis of nuclear ribosomal ITS DNA sequences show a strongly supported monophyletic group containing Philippine and Bornean species previously in Begonia sect. Diploclinium, and the type of Begonia sect. Baryandra, B. oxysperma. This clade forms the basis for the now much-expanded Begonia sect. Baryandra, which as defined here contains 49 species and has its centre of diversity in the Philippines.ConclusionsA natural classification for a much expanded Begonia sect. Baryandra has been provided. This paper highlights the feasibility of moving towards a natural classification of Asian Begonia step by step as information comes to light through building upon previous framework phylogenies with denser sampling.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-38) contains supplementary material, which is available to authorized users.

Systematic position of Cyathocline Cass. (Asteraceae): evidences from molecular, cytological and morphological data

Cyathocline, a small genus, has been treated as a member of subtribe Grangeinae of tribe Astereae (Asteraceae), but has been neglected in molecular phylogenetic analyses of Astereae. Plastid trnL-F and nuclear ribosomal DNA ITS sequences were used to carry out phylogenetic analyses of Cyathocline (represented by C. purpurea) through maximum parsimony and Bayesian analyses. In addition, its karyotype, morphology and micromorphology were also investigated. The results show that in our three phylogenetic trees, C. purpurea is deeply nested within the Blumea clade and/or the Inulinae clade, and is closest to Blumea balsamifera (Inuleae, Inulinae). C. purpurea is similar to Blumea in chromosome size bimodality and to Inulinae in one single large oxalate crystal within each cell of the cypsela epidermis, which, together with molecular evidences, suggests strongly that Cyathocline should be transferred from Astereae to Inuleae subtribe Inulinae. Although C. purpurea has many anomalous features, its most characters still are within a wide range of morphological variations of Blumea. DNA data and the karyotypic character support to merge C. purpurea into Blumea. As a result, the new combination Blumea purpurea (O. Kuntze) W.P. Li was made.