Sequence Analysis Of Clone Libraries Research Articles

Phylogenic diversity and tissue specificity of fungal endophytes associated with the pharmaceutical plant, Stellera chamaejasme L. revealed by a cultivation-independent approach.

The fungal endophytes associated with medicinal plants have been demonstrated as a reservoir with novel natural products useful in medicine and agriculture. It is desirable to explore the species composition, diversity and tissue specificity of endophytic fungi that inhabit in different tissues of medicinal plants. In this study, a culture-independent survey of fungal diversity in the rhizosphere, leaves, stems and roots of a toxic medicinal plant, Stellera chamaejasme L., was conducted by sequence analysis of clone libraries of the partial internal transcribed spacer region. Altogether, 145 fungal OTUs (operational taxonomic units), represented by 464 sequences, were found in four samples, of these 109 OTUs (75.2 %) belonging to Ascomycota, 20 (13.8 %) to Basidiomycota, 14 (9.7 %) to Zygomycota, 1 (0.7 %) to Chytridiomycota, and 1 (0.7 %) to Glomeromycota. The richness and diversity of fungal communities were strongly influenced by plant tissue environments, and the roots are associated with a surprisingly rich endophyte community. The endophyte assemblages associated with S. chamaejasme were strongly shaped by plant tissue environments, and exhibited a certain degree of tissue specificity. Our results suggested that a wide variety of fungal assemblages inhabit in S. chamaejasme, and plant tissue environments conspicuously influence endophyte community structure.

Primer pairs for the specific environmental detection and T-RFLP analysis of the ubiquitous flagellate taxa Chrysophyceae and Kinetoplastea

Bacterivorous protists play a key role in microbial soil food webs, however due to the lack of specific PCR protocols targeting selected protist taxa, knowledge on the diversity and dynamics of these groups is scarce. We developed specific PCR primers in combination with a T-RFLP protocol for the cultivation-independent analysis of two important taxa of bacterivorous flagellates, the Chrysophyceae and Kinetoplastea, in soil samples. Sequence analysis of clone libraries originating from two soils in temperate regions demonstrated the specificity of the respective primer pairs. Clone sequences affiliating to the Chrysophyceae mainly clustered within the clade C2, which has been known so far for its presence mainly in cold climatic regions, whereas Kinetoplastea sequences were mainly related to the Neobodonid clade. Based on an in silico restriction analysis of database sequence entries, suitable restriction enzymes for a T-RFLP approach were selected. This in silico approach revealed the necessity to use a combination of two restriction enzymes for T-RFLP analysis of the Chrysophyceae. Soil T-RFLP profiles reflected all T-RFs of the clone library sequences obtained from the same soils and allowed to distinguish flagellate communities from different sites. We propose to use these primer pairs for PCR detection and rapid fingerprint screening in environmental samples and envisage their use also for quantitative PCR or next generation sequencing approaches.

Anaerobic Biodegradation of Longer-Chain n-Alkanes Coupled to Methane Production in Oil Sands Tailings

Extraction of bitumen from mined oil sands ores produces enormous volumes of tailings that are stored in settling basins (current inventory ≥ 840 million m(3)). Our previous studies revealed that certain hydrocarbons (short-chain n-alkanes [C(6)-C(10)] and monoaromatics [toluene, o-xylene, m-xylene]) in residual naphtha entrained in the tailings are biodegraded to CH(4) by a consortium of microorganisms. Here we show that higher molecular weight n-alkanes (C(14), C(16), and C(18)) are also degraded under methanogenic conditions in oil sands tailings, albeit after a lengthy lag (~180 d) before the onset of methanogenesis. Gas chromatographic analyses showed that the longer-chain n-alkanes each added at ~400 mg L(-1) were completely degraded by the resident microorganisms within ~440 d at ~20 °C. 16S rRNA gene sequence analysis of clone libraries implied that the predominant pathway of longer-chain n-alkane metabolism in tailings is through syntrophic oxidation of n-alkanes coupled with CO(2) reduction to CH(4). These studies demonstrating methanogenic biodegradation of longer-chain n-alkanes by microbes native to oil sands tailings may be important for effective management of tailings and greenhouse gas emissions from tailings ponds.

Issues of anamorphic fungi

At the time when there is a move towards the acceptance of one name for one biological fungal species, Fungal Diversity documents the importance of anamorphic fungi with a special issue devoted to them. The present issue comprises 13 papers devoted to various topics concerning the anamorphic fungi with contributions on phylogeny, chemistry, ecology, post harvest importance, molecular detection and descriptions of some plant pathogens. The first paper is a review of the biogeography and phylogeography of Fusarium. This important paper questions several trends in the understanding of this important genus which causes a wide variety of plant diseases, produces a number of mycotoxins and is becoming increasingly recognized as a significant human pathogen. The authors look at several examples where surveys of non agro-systems question the present understanding of this extraordinary genus and must be read. The second paper is also a review of the chemical and bioactive producing capabilities of the remarkable genus Pestalotiopsis. This mostly endophytic genus is especially productive with regard to the accumulation of a diverse array of mostly bioactive compounds. More than 130 different compounds with antifungal, antimicrobial, and antitumor activities have been isolated from various species of this amazing genus. The third and fourth papers deal with post harvest topics. Colletotrichum gloeosporioides was previously reported to be the casual agent of anthracnose of most tropical fruits. This taxon, however, was recently epitypified and has been shown to be a species complex. A molecular study of isolates from Laos and Thailand causing anthracnose of eight tropical fruits shows that species other than C. gloeosporioides are responsible for anthracnose of most tropical fruits. This astounding result illustrates an urgent need to carry out research on re-inventory of tropical plant pathogens and should result in an unprecedented increase in phytopathogen research. Thirty one species belonging to 17 fungal genera were found to be associated with sorghum grain samples imported to the Kingdom of Saudi Arabia. These anamorphic fungi are important post harvest organisms producing important mycotoxins. The papers recommends that rigorous quarantine and healthy storage conditions should be undertaken to minimize fungal contamination and prevent further hazard to human and animal health. Papers five to seven deal with assessing fungal biodiversity from environmental samples using molecular analysis. Sette et al. profiled the fungal community structure found in a Brazilian energy transmission tower with signs of corrosion and/or biofilm formation using cloning (ITS-rRNA gene libraries), a culture-dependent technique. A total of 31 isolates comprising ten filamentous fungi and four yeasts were recovered from enrichment cultures showing the usefulness of this method. Klaubauf et al. were also successfully able to use RFLP and sequence analysis of clone libraries of the partial ITS/LSU-region as a culture-independent method to survey fungal diversity in four arable soils and one grassland in Lower Austria. Seena et al. show that aquatic hyphomycetes can be directly K. D. Hyde (*) School of Science, Mae Fah Luang University, Tasud, Chiang Rai 57100, Thailand e-mail: kdhyde2@gmail.com

Molecular diversity of fungal communities in agricultural soils from Lower Austria.

A culture-independent survey of fungal diversity in four arable soils and one grassland in Lower Austria was conducted by RFLP and sequence analysis of clone libraries of the partial ITS/LSU-region. All soils were dominated by the ascomycetous orders Sordariales, Hypocreales and Helotiales, taxa that are known from traditional cultivation approaches to occur in agricultural soils. The most abundant genus in the investigated soils was Tetracladium, a hyphomycete which has been described as occurring predominantly in aquatic habitats, but was also found in agricultural soils. Additionally, soil clone group I (SCGI), a subphylum at the base of the Ascomycota with so far no cultivated members, was identified at high frequency in the grassland soil but was below detection limit in the four arable fields. In addition to this striking difference, general fungal community parameters like richness, diversity and evenness were similar between cropland and grassland soils. The presented data provide a fungal community inventory of agricultural soils and reveal the most prominent species.

Molecular Characterization of Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Methanogenic Communities

Previous research demonstrated that methanogenic cultures enriched from Baltimore Harbor (Baltimore, MD) sediments were able to degrade naphthalene and phenanthrene. In this report, the degradation activity was maintained through a sequential transfer without adding additional sediments and the established polycyclic aromatic hydrocarbon (PAH)-degrading methanogenic communities were characterized via comparative sequence analysis of clone libraries of 16S rRNA genes amplified using bacteria-specific and Archaea-specific primers. The phylogenetic analysis indicated that the addition of PAHs clearly shifted the structure of the methanogenic community and resulted in an increase in populations of species previously found in other hydrocarbon-degrading communities. Of particular interest is the fact that the dominant microbial population of the naphthalene cultures was different from that of the phenanthrene cultures, suggesting that different species are involved in the degradation. Finally, this information may lead to the identification and isolation of methanogenic populations that can degrade PAHs.

Administration of Lactobacillus casei and Lactobacillus plantarum affects the diversity of murine intestinal lactobacilli, but not the overall bacterial community structure

Lactobacilli are normal inhabitants of the gastrointestinal tract (GIT) of many mammalian hosts. Their administration as probiotics in functional foods is currently a frequent practice, mainly because of their benefits to host health. It is therefore of interest to study the impact of administration of exogenous strains of Lactobacillus normally used as probiotics upon endogenous microbial populations. For this purpose, fecal and intestinal tissue samples were analyzed in a mouse model fed with a mixture of Lactobacillus plantarum and Lactobacillus casei isolated from commercially available dairy products. The murine intestinal microbiota was studied by means of cultivation-independent 16S rRNA gene-targeted techniques, namely denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of clone libraries. Multivariate statistical analysis was used to integrate datasets obtained from the different techniques applied. Whereas no differences were detected in the composition of the overall fecal bacterial community, changes were observed for intestinal tissue samples. Moreover, an increase in the diversity of gut lactobacilli was observed in fecal as well as intestinal tissue samples when mice received the mixture of L. casei and L. plantarum.