A polyphasic taxonomic study of Chryseobacterium strains isolated from dairy sources

A polyphasic taxonomic study, employing protein electrophoresis (SDS-PAGE), gas chromatographic analysis of cellular fatty acids (FAME), mol% G+C determination and DNA-DNA hybridizations, was undertaken on 103 dairy isolates shown to belong to Chryseobacterium. Reference strains of the Chryseobacterium species, CDC group IIb and Embedobacter brevis were included. SDS-PAGE analysis yielded good differentiation between the investigated species. About half of the strains could be clustered into nine major groups while the other half occupied a separate position. With FAME analysis no clear differentiation of the Chryseobacterium species (except C. meningosepticum) and SDS-PAGE groups could be achieved. FAME analysis, however, gave good differentiation between the Chryseobacterium and Empedobacter strains. The mol% G+C of the isolates tested, ranged between 36.4 and 39.0. The combination of SDS-PAGE and DNA-DNA hybridization identified a large group of dairy isolates as C. indologenes, one isolate as C. gleum and two new genotypic groups, comprising five and 15 dairy isolates respectively, emerged from the polyphasic study. Another large part of strains have a separate or uncertain position in Chryseobacterium and remained classified as Chryseobacterium species CDC group IIb.     

Degradation of the polyamine alkaloid aphelandrine by endophytic fungi isolated from Aphelandra tetragona

Members of the genus Aphelandra (Acanthaceae) produce rare macrocyclic polyamine alkaloids which consist of spermine acylated with two units of 3-(4-hydroxyphenyl)prop-2-enoic acid. Endophytic fungi were isolated from roots and shoots of Aphelandra tetragona and tested for their ability to metabolize the main alkaloid aphelandrine, which accumulates exclusively in the roots of the plants. Several endophytes were able to metabolize aphelandrine but only root endophytes belonging to the Nectriaceae were good metabolizers. In addition, the endophytes were grown on an agar medium containing putrescine, spermidine, or spermine as the sole nitrogen source. All fungi were able to grow on putrescine, but only the good aphelandrine metabolizers grew well on spermidine or spermine. Acremonium sp. 15, one of the most active metabolizers, grew also on a medium containing aphelandrine as sole nitrogen source. A number of strains thought to be conspecific with Acremonium sp. 15 were also tested for their ability to metabolize aphelandrine. The ability of the endophytes to metabolize aphelandrine suggests an ecological adaptation of the symbionts to their host. The possibility of using the aphelandrine metabolism as a taxonomic character is briefly discussed.     

A new meroterpenoid from endophytic fungus Talaromyces amestolkiae CS-O-1

Talaromyces amestolkiae CS-O-1, isolated from Tripterygium Wilfordii Hook. f., was identified based on its ITS and 18S rDNA gene sequencing. A new meroterpenoid, chrodrimanin T (1), along with six known compounds, nicotinamide (2), penipyridone D (3), penipyridone A (4), 3-benzylidene-8,8a-dihydroxy-2-methyl-hexahydro-pyrrolo[1,2-a]pyrazine-1,4-dione (5), butyl-isobutyl-phthalate (6), and aspergillumarin A (7), were isolated from Talaromyces amestolkiae CS-O-1. The structures and relative configurations of these compounds were established by the analysis of HRMS, 1D and 2D-NMR spectroscopy and the comparison with data in the literature. The compounds 2–6 were first isolated from Talaromyces genus. Herein, the chemotaxonomic significance of these compounds is described.     

Reduced perylenequinone derivatives from an endophytic Alternaria sp. isolated from Pinus ponderosa

The consecutive solvent extraction of endophytic Alternaria sp. (DC401) isolated from Pinus ponderosa followed by chromatographic techniques led to the isolation of five perylenequinone compounds and one dihydronaphthaquinone derivative, which include three new perylenequinones (1–3). The compounds were identified as 6-methoxy-3,6a,7,10-tetrahydroxy-4,9-dioxo-4,5,6,6a,6b,7,8,9-octahydroperylene (1), 3,6a,9,10-tetrahydroxy-7,8-epoxy-4-oxo-4,5,6,6a,6b,7,8,9-octahydroperylene (2), 6-methoxy-3,6a,9,10-tetrahydroxy-7,8-epoxy-4-oxo-4,5,6,6a,6b,7,8,9-octahydroperylene (3), 3,6a,7,10-tetrahydroxy-4,9-dioxo-4,5,6,6a,6b,7,8,9-octahydroperylene (altertoxin I) (4), 3,6a,7,10-tetrahydroxy-4,9-dioxo-4,6a,6b,7,8,9-hexahydroperylene (dehydroaltertoxin I) (5), and 7-chloroscytalone (6). Structure of compounds 1–6 was determined on the basis of detailed spectroscopic analysis, as well as by comparison with literature reports. The antileismanial, antimicrobial, antimalarial and in vitro cytotoxic activities of compounds 1–6 were evaluated.     

Raw starch-degrading enzyme from newly isolated strains of endophytic fungi

Four newly isolated strains of endophytic fungi namely Gibberella pulicaris, Acremonium sp., `Synnematous' sp. and Nodilusporium sp. were compared for their degradative activity on raw and gelatinized starches, substrate specificity and optimum pH. Results showed that the raw starch-degrading enzyme from Acremonium sp. had a broad activity towards both small and large granule size of raw starches while the enzyme from other strains showed high activity toward starches of smaller granule size. Analysis of the end product by TLC showed that enzyme from Gibberella pulicaris, Acremonium and Nodilusporium sp. hydrolysed raw sago starch to produce solely glucose but the enzyme of `Synnematous' sp. produced glucose and maltose. This revised version was published online in July 2006 with corrections to the Cover Date.     

A polyphasic approach for the differentiation of environmental Vibrio isolates from temperate waters

Climate change and marine traffic lead to changing species communities in the oceans. Due to increasing seawater temperatures, pathogenic Vibrio species could become significant even in temperate waters. We classified mesophilic Vibrio isolates from the German Bight (North Sea) using a polyphasic approach with special emphasis on Vibrio parahaemolyticus. Matrix-assisted laser desorption/ionization time-of-flight MS was used as a primary screen to classify isolates, 16S rRNA gene and rpoB gene sequencing to identify species. Potential V. parahaemolyticus isolates were screened for regulatory or virulence-related genes (toxR, tlh, tdh, trh). To investigate genomic diversity, we applied repetitive-sequence-based PCRs. Results were evaluated and methods compared using multivariate statistical analysis. Most isolates were classified as V. parahaemolyticus or Vibrio alginolyticus. Reliable differentiation between both species was achieved by rpoB sequencing and toxR detection. Among the fingerprinting methods, ERIC-PCR showed the highest discriminatory power, displaying three separated clusters. These clusters represent the species V. parahaemolyticus, V. alginolyticus and one group in between. The frequent detection of V. parahaemolyticus in the German Bight reveals the urgency for further monitoring. In this context, a polyphasic approach, such as defined in this study, is needed to differentiate populations of V. parahaemolyticus and V. alginolyticus.     

Natural products of Alternaria sp., an endophytic fungus isolated from Salvadora persica from Saudi Arabia

This study is to evaluate the potential of endophytic fungi of Salvadora persica for the production of bioactive compounds against pathogenic bacteria and fungi. Forty-two fungal isolates were obtained from 135 young and old stem and 125 root segments. Those 42 isolates representing ten fungi include: Trichoderma sp. (the most common), two species of Alternaria, Rhizopus arrhizus and 6 sterile mycelia. The ten fungi were grown in liquid culture and their crude extracts were tested against pathogenic bacteria and fungi. Nine crude extracts gave positive reactions against pathogenic bacteria of which Alternaria sp. (A8) was chosen further study. The fungal isolate was growing as sterile mycelium and was identified by phylogenetic analyses based on LSU rDNA sequence data and it might represent undescribed species of Alternaria. Sixty-two bioactive chemical compounds were identified from the ethyl acetate crude extracts of Alternaria sp., of which the following were recorded as major compounds in the active sub-fractions. These compounds showed strong antibacterial activity in combination.     

A polyphasic approach towards the identification of strains belonging to Lactobacillus acidophilus and related species

A set of 98 strains belonging to nine species of the Lactobacillus acidophilus rRNA-group have been analysed by SDS-PAGE of cellular proteins, RAPD-PCR and AFLP with fluorescently labeled primers in order to find improved methods for their identification. Strains of the following phenotypically highly similar species were examined: L. acidophilus, L. amylovorus, L. crispatus, L. johnsonii, L. gasseri, L. gallinarum, L. helveticus, L. iners and L. amylolyticus. Although the majority of the species can be differentiated by SDS-PAGE of whole-cell proteins, the latter technique showed poor discrimination between L. gasseri and L. johnsonii strains and between some strains of L. amylovorus and L. gallinarum. However, this study shows that the RAPD-PCR (using at least 3 different primers followed by numerical analysis of the combined patterns) and AFLP are most suitable genomic fingerprinting techniques for the differentiation of all the species listed above, and that databases for identification can be constructed, particularly when commercially available molecular tool-kits are used. The separate species status of the recently described L. amylolyticus and L. iners was fully confirmed.     

New insights into the regulation of phytochelatin biosynthesis in A. thaliana cells from metabolite profiling analyses

In higher plants and some fungi, heavy metals induce the synthesis of chelating peptides known as phytochelatins (PCs). They are characterized by the general structure (gamma-Glu-Cys)n-Gly, but in some plant species, the C-terminal glycine can be replaced by serine, glutamine, glutamate or alanine, leading to iso-phytochelatins (iso-PCs). Although the distribution of iso-PCs is considered to differ from one species to another, we previously showed that Arabidopsis thaliana (A. thaliana) cells are able to synthesize most PC-related peptides (PCs and iso-PCs) described in the literature. We also observed an accumulation of the dipeptide gamma-glutamylcysteine (gamma-EC) when cadmium (Cd) (200 microM) was added to the culture medium, suggesting that either glutathione synthetase or glycine availability could be a limiting factor for the biosynthesis of PC-related peptides. In this context, the aim of the present work was to seek new insights into the regulation of PC synthesis by performing metabolic profiling using liquid chromatography-mass spectrometry. The levels of PC-related peptides and their precursors were measured in A. thaliana cells following Cd exposure. A range of doses (0, 50, 200 and 400 microM CdNO3) and kinetic studies (from 1 to 48 h) showed a dose threshold (50 microM CdNO3) and a lag time between the appearance of PCs and iso-PCs concomitant with the gamma-EC accumulation induced by Cd, occurring at cadmium concentrations above 50 microM. This accumulation was suppressed by supplementation of the culture medium with 25 mM glycine. Glycine supplementation had a limited impact on the concentrations of glutathione and PCs whereas the levels of most iso-PCs were significantly increased. Taken together, these results indicate that GSH is involved in the biosynthesis of the iso-PCs in vivo, and that the biosynthesis of PC-related peptides is limited by the availability of glycine in the presence of high cadmium concentrations.     

Altenusin, a biphenyl isolated from the endophytic fungus Alternaria sp., inhibits trypanothione reductase from Trypanosoma cruzi

Parasitic protozoan species belonging to the genera Trypanosoma and Leishmania are the etiological agents of several diseases in tropical areas of the world, for which there is an urgent need for effective and affordable treatment. In this regard, we are screening the Brazilian biodiversity, especially its flora and mycota, for natural products that could serve as leads for drug development against these diseases. Trypanothione reductase (TR) is an enzyme involved in the protection of Trypanosoma and Leishmania species against oxidative stress, and is considered to be a validated drug target. The endophytic fungus Alternaria sp. (UFMGCB55) was isolated from the plant Trixis vauthieri DC (Asteraceae), known to contain trypanocidal compounds. The organic extract of the culture of Alternaria sp. was able to inhibit TR by 99%, when tested at 20 microg mL(-1). Fractionation of the extract identified altenusin, a biphenyl derivative with an IC50 value of 4.3+/-0.3 microM in the TR assay. This compound is the first in its class to have shown TR inhibitory activity, opening new perspectives for the design of more effective derivatives that could serve as drug leads for new chemotherapeutic agents to treat trypanosomiasis and leishmaniasis.     

Metabolomic study reveals a selective accumulation of l-arginine in the d-ornithine treated tobacco cell suspension culture

The non-protein amino acid ornithine (Orn) plays essential roles in regulation of the urea cycle and polyamines biosynthesis in tobacco. Herein, we show that d-enantiomer of Orn, can actively participate in metabolites production in tobacco cells, functioning a positive role in plant cells metabolism, as opposed to the common l-enantiomer. Using a comprehensive amino acids and amines profiling method by liquid chromatography–electrospray ionization triple quadruple mass spectrometry (LC–ESI-QqQ-MS) in combination with chiral LC–ESI-MS, it was shown that d-Orn has a potential advantage in promoting selective and large accumulation of l-arginine (l-Arg) in tobacco cells. Exogenous d-Orn resulted in a selective up-regulation of l-Arg by 80-fold, while l-Orn slightly increased the levels of all amino acids. Changes of all the urea cycle related intermediates, e.g. citrulline, Arg and Orn were also shown to be critical following change of Orn's stereochemistry. GC/MS profiling of the metabolites revealed that high nicotine production was the dominant change driven by l-Orn treatments. From these observations, d-Orn was shown to be a selective regulator of l-Arg biosynthesis and the urea cycle. We propose that d-Orn has a potential function in the tobacco cells which through some previously unidentified mechanism result in l-Arg accumulation.     

Molecular assessment of diversity among endophytic diazotrophs isolated from subtropical Indian sugarcane

Twenty-two endophytic bacterial isolates from the roots of sugarcane were compared morphologically, biochemically and genetically. Gram staining, colony pigment, texture and other cultural characteristics were taken for morphological characterization. Oxidation-fermentation tests for D-glucose and D-sucrose, production of acid and hydrogen from different carbon source, oxidase activity, antibiotic and drug resistance patterns were chosen as the biochemical and physiological criteria. Twelve random decamer primers were used to analyze and compare these isolates through RAPD among themselves as well as with known standard diazotrophic strains. The isolates were compared through dendrograms constructed on the basis of similarity patterns obtained from biochemical and RAPD analysis. The estimated diversity through RAPD analysis was more evident than the diversity on the basis of morphological and biochemical characters. Within Acetobacter group, the isolates showed substantial genetic diversity for future exploitation as PGPRs and diazotrophic associative endophytes.     

Antifungal activity of altenusin isolated from the endophytic fungus Alternaria sp. against the pathogenic fungus Paracoccidioides brasiliensis

BackgroundAltenusin is a biphenyl derivative isolated from different species of fungi, which presents several biological activities.AimsWe report the antifungal activity of the altenusin isolated from the endophytic fungus Alternaria sp., against clinical isolates of Paracoccidioides brasiliensis, and its action on cell walls of P. brasiliensis and the nonpathogenic yeast Schizosaccharomyces pombe.MethodsIn vitro antifungal activity of altenusin was evaluated using the broth microdilution method against 11 strains of P. brasiliensis and one strain of S. pombe. The effects of the altenusin on the cell wall were estimated using the sorbitol protection assay.ResultsThe altenusin presented strong activity against P. brasiliensis with MIC values ranging between 1.9 and 31.2 μg/ml, and 62.5 μg/ml for S. pombe. Our results demonstrated that the MIC values for altenusin were increased for P. brasiliensis Pb18 and for S. pombe when the medium was supplemented with sorbitol. Additionally, S. pombe cells treated with altenusin were more rounded in shape than untreated cells.ConclusionsAltenusin showed activity against clinical strains of P. brasiliensis at the concentration tested, and this compound probably affects fungal cell walls. These findings suggest that altenusin could act through the inhibition of cell wall synthesis or assembly in P. brasiliensis and S. pombe, and could be considered as a lead compound for the design of new antifungals.     

A polyphasic approach for the taxonomy of cyanobacteria: principles and applications

Taxonomic classification is the only method for understanding and exploring knowledge about organismal diversity. However, it is complicated in prokaryotic, phylogenetically old, phototrophic cyanobacteria, which contain very simple unicellular forms up to multicellular types with a differentiated and diversified thallus. Their cells are cytologically relatively simple, but variable in shape. Various genotypes are adaptable to various specialized ecosystems. The introduction and combination of modern molecular, cytomorphological and ecological methods in the taxonomy of cyanobacteria is necessary and should be accepted as the only method for the elaboration of their modern systematics. The combination of different methods should be based on molecular sequencing as the basic approach, to which must be added other criteria (morphological, ecological) if they are available and which are distinct and recognizable in cyanobacterial populations. The use of such characteristics is necessary and must be obligatorily included for the final characterization both of strains and natural populations. Application of this polyphasic, i.e. combined approach is considered as a unique, modern, unambiguous, unequivocal and a fully acceptable methodological procedure, but it is not yet commonly used, nor possible for all known cyanobacterial populations. The main principles and recent problems of this modern classification method are discussed in the following review and will be the basis of further discussion.     

Molecular characterization of Alternaria alternata population isolated from Upper Egyptian tomato fruits

Alternaria alternata is the most common fungal pathogen of tomatoes in Upper Egypt. Morphological identification of this fungus is challenging; therefore, this study searched for new classification tools based on molecular techniques. Using a dilution plating method, 67 strains of A. alternata were isolated from 34 samples of rotten tomato fruits representing the Giza 80 and Edkawy cultivars. The collected strains were identified using the amplification products of the internal transcribed spacer (ITS) region, glyceraldehyde 3‐phosphate dehydrogenase (Gpd) and Alt a1, which is a gene involved in the production of most of the allergens produced by A. alternata. The screening revealed that A. alternata constituted more than half of the total fungi recovered from rotten tomatoes in this study. According to the phylogenetic analysis using these three loci, the collected strains clustered in accordance with the host cultivar type from which they had been isolated. Specific gene random primer polymerase chain reaction (SGRP‐PCR) techniques indicated that the A. alternata population in the tested region has a high genetic diversity. The pathogenicity test showed that most of the A. alternata isolates (67.2%) were highly pathogenic, and no correlation was found between the phylogenetic analysis and pathogenicity. In addition, the influence of the fungicide Disan 80% on the collected strains showed significant differences that were attributed to the source of isolation.     

Molecular characterization of endophytic strains of Fusarium verticillioides (= Fusarium moniliforme) from maize (Zea mays. L)

The species Fusarium verticillioides (= F. moniliforme) is often found in maize seeds, constituting an important source of inoculum in the field. Fusarium spp., associated with symptomatic and asymptomatic plants, may be a primary causal agent of disease, a secondary invader or an endophyte. In the present work, endophytic fungi were isolated from two populations of Zea mays (BR-105 and BR-106) and their respective inbred lines. Within different inbred lines of maize, Fusarium was found at a frequency of 0 to 100% relative to the number of total isolated fungi. The frequency with which the genus occurred was practically the same in the two field sites (around 60%). Twenty-one F. verticillioides strains were analysed using the random amplified polymorphic DNA (RAPD) technique, employing 10 random primers. Variability analysis of endophytic isolates via RAPD showed genome polymorphism taxa of species around 60%. Endophytic isolates were clustered by their sites of origin. RAPD analysis clustered the endophytic isolates by their maize inbred lines hosts (Mil-01 to Mil-06), whereas at site A they clustered into two major groups related to the maize gene pool (BR-105 or BR-106 population). All strains isolated from seeds collected in Site A, except strains L9 and L10, were sub-grouped according to maize inbred lines. The analysis showed a discrete sub-grouping at site B. Results obtained here could be explained by a co-evolution process involving endophytic isolates of F. verticillioides and maize inbred lines.     

Chemical constituents isolated from the roots and rhizomes of Smilacina japonica

Phytochemical investigation of the roots and rhizomes of Smilacina japonica A. Gray led to the isolation of 16 compounds, including six steroidal saponins (1-6), one amide (7), one fatty alcohol (8), one steroidal sapogenin (9), two flavones (10-11), one glycoside (12), one phenol (13), one aliphatic acid (14), and two sterols (15-16). All of these compounds were isolated for the first time from the roots and rhizomes of S. japonica while compounds 1-13 were identified for the first time from the genus Smilacina, of which compounds 9, 11, and 13 were isolated from the Liliaceae family for the first time. Furthermore, the isolation of compound 9 was reported for the first time in plants. Their structures were identified by spectroscopic methods and compared previously published data. The chemotaxonomic significance of these isolated compounds has also been discussed.     

Optimization of protease production by endophytic fungus,Alternaria alternata,isolated from an Australian native plant

Endophytes are recognised as potential sources of novel secondary metabolites, including enzymes and drugs, with applications in medicine, agriculture and industry. There is a growing need for new enzymes, including proteases, for use in industry that can function under a variety of conditions. In this study, three fungal endophytes (Alternaria alternata, Phoma herbarum and an unclassified fungus), were isolated from the Australian native plant, Eremophilia longifolia, and assessed for production of proteases. The lyophilised growth media obtained after fungal fermentation were analysed for protease production using enzyme activity assays. Protease production was optimised by assessing the effects of temperature, pH, carbon source and nitrogen source on activity. A. alternata showed the greatest protease activity in a wide range of pH (3–9). The broadest activity between 9 and 50 °C was observed at pH 7, suggesting a neutral protease. Overall, the optimum conditions were 37 °C and pH 7 with a maximum specific activity value of 69.86 BAEE units/mg. The characteristics demonstrated by this fungal endophyte showed that it is a potential source of an enzyme with particular application in the dairy industry. However, further studies of the tolerance to higher temperatures and pH will indicate whether the enzyme is suitable to such applications.