Chemical constituents and their antibacterial activity from the tropical endophytic fungus Diaporthe sp. F2934.

AIMS: To isolate, characterize and determine the antibacterial activities of compounds produced by the endophytic fungus Diaporthe sp. F2934, cultivated on malt extract agar. METHODS AND RESULTS: The fungus was cultivated aseptically in Petri dishes containing malt extract agar at 25°C for 15 days. Crude extract was obtained from mycelium using ethyl acetate and sonication, and was fractioned using classic chromatography and HPLC. The structures of phomosines and chromanones were established by NMR experiments including HMQC, HMBC and COSY. Their molecular formulas were determined by ESI-TOFMS. We obtained six compounds: (1) 4H-1-benzopyra-4-one-2,3-dihydro-5-hydroxy-2,8-dimetyl, (2) 4H-1-benzopyran-4-one-2,3-dihydro-5-hydroxy-8-(hydroxylmethyl)-2-methyl, (3) 4H-1-benzopyra-4-one-2,3-dihydro-5-methoxyl-2,8-dimetyl, (4) phomosine A, (5) phomosine D and (6) phomosine C. Isolated compounds 1, 2 and 5 were inactive against 15 micro-organisms, but phomosines A and C were active against diverse Gram-negative and Gram-positive bacteria. CONCLUSIONS: A group of new chromanones and known phomosines have been isolated from the genus Diaporthe (Diaporthe sp. F2934). The results obtained confirm the wide chemical diversity produced by endophytic fungi, specifically the genus Diaporthe. In addition, phomosines A and C may be considered as antimicrobial agents that can be used to guide the development of new antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Our phylogenetic analysis places Diaporthe sp. F2934 as sister to the Diaporthe cynaroidis clade. Three chromanones were isolated and identified, for the first time, using crude extract obtained from Diaporthe F2934. From this extract phomosines A, C and D were also purified. Regarding Staphylococcus aureus, the inhibition zone diameter (IZD) for phomosine A was 20% higher than the standard drug, vancomycin. When cultivated as described here, Diaporthe sp. F2934 produced new and antimicrobial compounds.

Chemical and bioactive natural products from Microthyriaceae sp., an endophytic fungus from a tropical grass.

UNLABELLED: In screening for natural products with antiparasitic activity, an endophytic fungus, strain F2611, isolated from above-ground tissue of the tropical grass Paspalum conjugatum (Poaceae) in Panama, was chosen for bioactive principle elucidation. Cultivation on malt extract agar (MEA) followed by bioassay-guided chromatographic fractionation of the extract led to the isolation of the new polyketide integrasone B (1) and two known mycotoxins, sterigmatocystin (2) and secosterigmatocystin (3). Sterigmatocystin (2) was found to be the main antiparasitic compound in the fermentation extract of this fungus, possessing potent and selective antiparasitic activity against Trypanosoma cruzi, the cause of Chagas disease, with an IC50 value of 0.13 µmol l(-1) . Compounds 2 and 3 showed high cytotoxicity against Vero cells (IC50 of 0.06 and 0.97 µmol l(-1) , respectively). The new natural product integrasone B (1), which was co-purified from the active fractions, constitutes the second report of a natural product possessing an epoxyquinone with a lactone ring and exhibited no significant biological activity. Strain F2611 represents a previously undescribed taxon within the Microthyriaceae (Dothideomycetes, Ascomycota). SIGNIFICANCE AND IMPACT OF THE STUDY: The present study attributes new antiparasitic and psychoactive biological activities to sterigmatocystin (2), and describes the structure elucidation of the new natural product integrasone B (1), which possesses a rare epoxyquinone with a lactone ring moiety. This is also the first report of sterigmatocystin (2) isolation in a fungal strain from this family, broadening the taxonomic range of sterigmatocystin-producing fungi. The study also presents taxonomic analyses indicating that strain F2611 is strongly supported as a member of the Microthyriaceae (Ascomycota), but is not a member of any previously known or sequenced genus.

Do the antiherbivore traits of expanding leaves in the Neotropical tree Inga paraensis (Fabaceae) vary with light availability?

Treefall gaps in tropical forests have a profound effect on plants growing in the understory, primarily due to increased light availability. In higher light, mature leaves typically have increased anti-herbivore defenses. However, since the majority of herbivory occurs while leaves are expanding, it is important to determine whether defense expression during the short period of leaf expansion is canalized (invariant) or plastic in response to variation in light. Therefore, we examined young leaves of Inga paraensis (Fabaceae) saplings growing along a light gradient in a terra-firme forest in Central Amazonia. We quantified leaf production and expansion time, dry mass of phenolics, saponins, and nitrogen, ants attracted to extrafloral nectaries, and leaf consumption. Over the entire light gradient, the number of leaves produced per flush increased by 50 % and the mass of phenolic compounds by 20 %, but no other traits changed. On average, 39 % of leaf area was consumed with no difference across the light gradient. Alone, none of the leaf traits was a significant predictor of leaf consumption, except for phenolics, which showed a positive relationship. Multiple regressions showed that leaf consumption was positively related to more leaves per flush and a higher concentration of phenolics in leaves. Unlike studies of mature leaves, young leaves of I. paraensis show low plasticity in defense traits across a light gradient, suggesting that leaf development is canalized.

Is extrafloral nectar production induced by herbivores or ants in a tropical facultative ant-plant mutualism?

Many plants use induced defenses to reduce the costs of antiherbivore defense. These plants invest energy in growth when herbivores are absent but shunt energy to defense when herbivores are present. In contrast, constitutive defenses are expressed continuously regardless of herbivore presence. Induction has been widely documented in temperate plants but has not been reported from tropical plants. Most tropical plants have higher, more constant herbivore pressure than temperate plants. In this situation, it is hypothesized that constitutive defenses rather than induced defense would be favored. Using natural herbivores of four species of Inga saplings on Barro Colorado Island, Panama, herbivore presence was crossed with ant presence to determine their effects on extrafloral nectar production. Analysis of nectar samples revealed that Inga species do not induce nectar production in response to herbivores. This result is not due to an inability of the plants to respond, as the plants in this study increased nectar production in response to light and ant presence. Contrary to most induction experiments with temperate ecosystem plants, these results demonstrate that tropical plants do not induce one type of defense, and they suggest that the most adaptive defense strategies are different for the two ecosystems.

Host specificity of Lepidoptera in tropical and temperate forests.

For numerous taxa, species richness is much higher in tropical than in temperate zone habitats. A major challenge in community ecology and evolutionary biogeography is to reveal the mechanisms underlying these differences. For herbivorous insects, one such mechanism leading to an increased number of species in a given locale could be increased ecological specialization, resulting in a greater proportion of insect species occupying narrow niches within a community. We tested this hypothesis by comparing host specialization in larval Lepidoptera (moths and butterflies) at eight different New World forest sites ranging in latitude from 15 degrees S to 55 degrees N. Here we show that larval diets of tropical Lepidoptera are more specialized than those of their temperate forest counterparts: tropical species on average feed on fewer plant species, genera and families than do temperate caterpillars. This result holds true whether calculated per lepidopteran family or for a caterpillar assemblage as a whole. As a result, there is greater turnover in caterpillar species composition (greater beta diversity) between tree species in tropical faunas than in temperate faunas. We suggest that greater specialization in tropical faunas is the result of differences in trophic interactions; for example, there are more distinct plant secondary chemical profiles from one tree species to the next in tropical forests than in temperate forests as well as more diverse and chronic pressures from natural enemy communities.

Causes and consequences of monodominance in tropical lowland forests.

Tropical canopy dominance in lowland, well-drained forests by one plant species is a long-standing conundrum in tropical biology. Research now shows that dominance is not the result of one trait or mechanism. We suggest that the striking dominance of Gilbertiodendron dewevrei in the Ituri Forest of northeastern Congo is the result of a number of traits in adult trees that significantly modify the understory environment, making it difficult for other species to regenerate there. Adults cast deep shade that reduces light levels in the understory of the Gilbertiodendron forest to levels significantly lower than in the mixed-species forest. Moreover, the monodominant forest has deep leaf litter that could inhibit the establishment of small-seeded species, and the leaf litter is slow to decompose, potentially causing the low availability of nitrogen. We expect that juveniles of Gilbertiodendron may have an advantage in this environment over other species. In general, it appears that all tropical monodominant species share a similar suite of traits.

Contrasting modes of light acclimation in two species of the rainforest understory.

In tropical rainforests, the increased light associated with the formation of treefall gaps can have a critical impact on the growth and survivorship of understory plants. Here we examine both leaf-level and whole-plant responses to simulated light gap formation by two common shade-tolerant shrubs, Hybanthus prunifolius and Ouratea lucens. The species were chosen because they differed in leaf lifespans, a trait that has been correlated with a number of leaf- and plant-level processes. Ouratea leaves typically live about 5 years, while Hybanthus leaves live less than 1 year. Potted plants were placed in the understory shade for 2 years before transfer to a light gap. After 2 days in high light, leaves of both species showed substantial photoinhibition, including reduced CO2 fixation, F v/F m and light use efficiency, although photoinhibition was most severe in Hybanthus. After 17 days in high light, leaves of both species were no longer photoinhibited. In response to increased light, Ouratea made very few new leaves, but retained most of its old leaves which increased photosynthetic capacity by 50%. Within a few weeks of transfer to high light, Hybanthus had dropped nearly all of its shade leaves and made new leaves that had a 2.5-fold greater light-saturated photosynthetic rate. At 80 days after transfer, the number of new leaves was 4.9-fold the initial leaf number. After 80 days in high light, Hybanthus had approximately tenfold greater productivity than Ouratea when leaf area, photosynthetic capacity, and leaf dark respiration rate were all taken into account. Although both species are considered shade tolerant, we found that their growth responses were quite different following transfer from low to high light. The short-lived Hybanthus leaves were quickly dropped, and a new canopy of sun leaves was produced. In contrast, Ouratea showed little growth response at the whole-plant level, but a greater ability to tolerate light stress and acclimate at the leaf level. These differences are consistent with predictions based on leaf lifespan and are discussed within the context of other traits associated with shade-tolerant syndromes.

Extraction of phenolic compounds from fresh leaves: A comparison of methods.

The conventional sonicator/shaker bath method for phenolic extraction was compared with a less traditional one using a homogenizer. The homogenizer proved to be both more efficient and consistent in extracting phenolics from tender, as well as tough, leaves. We propose that adoption of the homogenizer technique will increase phenolic yield and efficiency.

Genomic structure and sequence variation of a 3.3-kb repeat DNA element of the kangaroo rat, Dipodomys ordii.

The genomic structure and sequence variation of a 3.3-kb repeat DNA element, representing 5% of the genome of the kangaroo rat Dipodomys ordii, has been investigated. Most of the repeats are arranged in tandem arrays of 50 kb or more. Thirteen randomly selected genomic clones have been mapped with twelve restriction enzymes. The frequency of sequence divergence in the genomic clones is 0.5%. The clone maps and the genomic structure studies have permitted the characterization of a number of variant members of the 3.3-kb repeat family. The genomic organization of the repeat resembles that for repeated DNAs found in large tandem arrays or satellites.

Novel Phycoerythrins in Marine Synechococcus spp. : Characterization and Evolutionary and Ecological Implications.

Four clones of the marine, unicellular, cyanobacteria Synechococcus spp., were examined for the spectral and biochemical features of their phycoerythrins (PE) and their photosynthetic characteristics. Two spectral types of PE which are distinct from known PEs were found. One PE type possessed absorption maxima at 500 and 545 nm and a fluorescence emission at 560 nm. Upon denaturation in acid-urea, two chromophore absorption maxima were obtained, one corresponding to phycourobilin (A(max) 500 nm) and one at 558 nm, ascribed to a phycoerythrobilin-like chromophore. The ratio of phycoerythrobilin-like to phycourobilin chromophores was 4.9:1.3. This PE possessed two subunits of M(r)s of 17.0 and 19.5 kD for the alpha and beta subunits, respectively. The other PE possessed a single symmetrical absorption at 551 nm and a fluorescence emission at 570 nm. This phycobiliprotein showed a single chromophore absorption band (A(max) 558 nm) and yielded two polypeptides, an alpha of 17.5 kD and a beta subunit of 20.8 kD. Both PEs showed a (alpha, beta)(n) structure. The presence of phycoerythrobilin-like chromophores (A(max) 558 nm) appears to be diagnostic of this marine cyanobacterial group. The features of these PEs combined with additional biochemical data, suggest a possible evolutionary link between the PE-containing marine Synechococcus group and the red algal chloroplast. When the Synechococcus clones were grown under low light intensity the PE-containing clones showed higher photosynthetic performance, larger photosynthetic units sizes, reaction center I to II ratios near unity, and steeper initial slopes of photosynthesis versus irradiance curves than a non-PE-containing clone. These findings demonstrate the high photosynthetic efficiency of PE-containing clones in low light environments common to middepth neritic and oceanic habitats.

Light-Harvesting System of the Red Alga Gracilaria tikvahiae: II. Phycobilisome Characteristics of Pigment Mutants.

Phycobilisomes were isolated from wild type Gracilaria tikvahiae and a number of its genetically characterized Mendelian and non-Mendelian pigment mutants in which the principal lesions result in an increase or decrease in the accumulation of phycoerythrin. Both the size and phycoerythrin content of the phycobilisomes are proportional to the phycoerythrin content of the crude algal extracts. In most of the strains examined, the structure and function of the phycocyanin-allophycocyanin phycobilisome cores are the same as in wild type. The phycobilisome architecture is derived from wild type by the addition or removal of phycoerythrin. The same pattern is observed for the phycobilisome of mos(2) which contains a large excess of phycocyanin that is not bound to the phycobilisome. The single exception is a yellow, non-Mendelian mutant, NMY-1, which makes functional phycobilisomes composed of phycoerythrin and allophycocyanin with almost no phycocyanin. Characterization of the ;linker' polypeptides of the phycobilisome indicates that a 29 kilodalton protein is required for the stable incorporation of phycocyanin into the phycobilisome. Evidence is provided for the requirement of nuclear and cytoplasmic genes in phycobilisome synthesis and assembly. The symmetry properties of the phycobilisome are considered and a structural model for the reaction center II-phycobilisome organization is presented.

Light-Harvesting System of the Red Alga Gracilaria tikvahiae: I. Biochemical Analyses of Pigment Mutations.

Wild type Gracilaria tikvahiae, a macrophytic red alga, and fourteen genetically characterized pigment mutants were analyzed for their biliprotein and chlorophyll contents. The same three biliproteins, phycoerythrin, phycocyanin, and allophycocyanin, which are found in the wild type are found in all the Mendelian and non-Mendelian mutants examined. Some mutants overproduce R-phycoerythrin while others possess only traces of phycobiliprotein; however, no phycoerythrin minus mutants were found. Two of the mutants are unique; one overproduces phycocyanin relative to allophycocyanin while the nuclear mutant obr synthesizes a phycoerythrin which is spectroscopically distinct from the R-phycoerythrin of the wild type. The phycoerythrin of obr lacks the typical absorption peak at 545 nanometers characteristic of R-phycoerythrin and possesses a phycoerythrobilin to phycourobilin chromophore ratio of 2.6 in contrast to a ratio of 4.2 found in the wild type. Such a lesion provides evidence for the role of nuclear genes in phycoerythrin synthesis. In addition, comparisons are made of the pigment compositions of the Gracilaria strains with those of Neoagardhiella bailyei, a macrophytic red alga which has a high phycoerythrin content, and Anacystis nidulans, a cyanobacterium which lacks phycoerythrin. The mutants described here should prove useful in the study of the genetic control of phycobiliprotein synthesis and phycobilisome structure and assembly.

Photosynthetic Unit Organization in a Red Alga : Relationships between Light-Harvesting Pigments and Reaction Centers.

The relative concentration of biliproteins, phycobilisomes, chlorophyll a, and reaction centers I and II are reported for Neoagardhiella bailyei, a macrophytic red alga collected in the field and compared with Anacystis nidulans, a cyanobacterium cultured in the laboratory. The ratios of chlorophyll to reaction center I, of chlorophyll to reaction center II, and the mass of phycobiliprotein per reaction center II are quite similar in Neoagardhiella and Anacystis, supporting the concept that the red algal chloroplast is derived from a cyanobacterial progenitor. The ratios of reaction center I to reaction center II are about 2.3 in both species. The Anacystis phycobilisome has about 40% of the mass of the Neoagardhiella phycobilisome, 4.9 and 14.9 x 10(6) daltons, respectively. The reaction center II/phycobilisome ratio is about 1.7 in Anacystis and 4.1 in Neoagardhiella. Phycobilisome size and physical restrictions on phycobilisome packing may be a major constraint on the reaction center II-phycobilisome association and the assembly of the photosynthetic membrane in both the red algae and cyanobacteria.

Morphology of a novel cyanobacterium and characterization of light-harvesting complexes from it: Implications for phycobiliprotein evolution.

The morphology of the marine cyanobacterium DC-2 and two light-harvesting complexes from it have been characterized. DC-2 has an outer cell wall sheath not previously observed, the purified phycoerythrin shows many unusual properties that distinguish it from all phycoerythrins characterized to date, and isolated phycobilisomes have a single absorption band at 640 nm in the phycocyanin-allophycocyanin region of the spectrum. On the basis of these observations we suggest that DC-2, rather than being a member of the Synechococcus group, should be placed in its own taxonomic group. In addition, the particular properties of the isolated phycoerythrin suggest that it may be representative of an early stage in the evolution of the phycoerythrins. These observations are of special interest in light of the contribution DC-2 and related cyanobacteria may make to global primary productivity.