Changes in secondary metabolism during stromatal ontogeny of Hypoxylon fragiforme

Stromata of Hypoxylon fragiforme were studied during the vegetation period by hplc profiling, revealing changes in the composition during stromatal development. Cytochalasin H and two new cytochalasins named fragiformins A–B were identified as major constituents of the young, maturing stromata, whereas mature, ascogenous material yielded large amounts of mitorubrin-type azaphilones. The above compounds, further cytochalasins from Xylariaceae and other fungi, and additional azaphilones of the mitorubrin type were assayed for their nematicidal effects against Caenorhabditis elegans and their antimicrobial activities against Bacillus subtilis, Yarrowia lipolytica, and various filamentous fungi. The results confirmed data in the literature on broad-spectrum non-selective activities of azaphilones and cytochalasins in biological systems. Most interestingly, laboratory cultures of the above Hypoxylon spp. mainly produced dihydroisocoumarin derivatives and were found devoid of mitorubrins and cytochalasins. These rather drastic changes in the secondary metabolism of H. fragiforme and the above biological activities are discussed in relation to the possible biological functions of secondary metabolites (extrolites) in the Hypoxyloideae.     

Resurrection and emendation of the Hypoxylaceae,recognised from a multigene phylogeny of the Xylariales

A multigene phylogeny was constructed, including a significant number of representative species of the main lineages in the Xylariaceae and four DNA loci the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, the second largest subunit of the RNA polymerase II (RPB2), and beta-tubulin (TUB2). Specimens were selected based on more than a decade of intensive morphological and chemotaxonomic work, and cautious taxon sampling was performed to cover the major lineages of the Xylariaceae; however, with emphasis on hypoxyloid species. The comprehensive phylogenetic analysis revealed a clear-cut segregation of the Xylariaceae into several major clades, which was well in accordance with previously established morphological and chemotaxonomic concepts. One of these clades contained Annulohypoxylon, Hypoxylon, Daldinia, and other related genera that have stromatal pigments and a nodulisporium-like anamorph. They are accommodated in the family Hypoxylaceae, which is resurrected and emended. Representatives of genera with a nodulisporium-like anamorph and bipartite stromata, lacking stromatal pigments (i.e. Biscogniauxia, Camillea, and Obolarina) appeared in a clade basal to the xylarioid taxa. As they clustered with Graphostroma platystomum, they are accommodated in the Graphostromataceae. The new genus Jackrogersella with J. multiformis as type species is segregated from Annulohypoxylon. The genus Pyrenopolyporus is resurrected for Hypoxylon polyporus and allied species. The genus Daldinia and its allies Entonaema, Rhopalostroma, Ruwenzoria, and Thamnomyces appeared in two separate subclades, which may warrant further splitting of Daldinia in the future, and even Hypoxylon was divided in several clades. However, more species of these genera need to be studied before a conclusive taxonomic rearrangement can be envisaged. Epitypes were designated for several important species in which living cultures and molecular data are available, in order to stabilise the taxonomy of the Xylariales.     

Macrocarpones,novel metabolites from stromata of <Emphasis Type="Italic">Hypoxylon macrocarpum</Emphasis>, and new evidence on the chemotaxonomy of <Emphasis Type="Italic">Hypoxylon</Emphasis> species

Several species of the genus Hypoxylon (Xylariaceae) were studied for morphological characters and HPLC-based secondary metabolite profiles. It was confirmed that Hypoxylon is divided into two groups of species, containing either Mitorubrin type azaphilones or binaphthyls, respectively, as main metabolites. In Hypoxylon species of the latter group, some metabolites that are known from the allied genus Daldinia (i.e., Daldinal A in H. fuscum and Daldinin C in H. fuscopurpureum), were encountered for the first time. Moreover, three novel aromatic polyketides, for which the trivial names Macrocarpones A, B and C are proposed, were isolated from ascostromata of Hypoxylon macrocarpum and identified by high resolution mass spectrometry and 2D-NMR spectroscopy. Orsellinic Acid was also identified from ascostromata of H. howeianum and detected by HPLC-MS and HPLC-UV/Vis fingerprinting methodology in H. fragiforme and H. rubiginosum, but not in other examined Hypoxylon species. Due to these studies, H. fuscopurpureum sensu Y.-M. Ju & J.D. Rogers 1996 was identified as a new record for Germany.     

Numerical taxonomy of some species of hypoxylon

Numerical analysis has resulted in a division of the coloured and non-coloured species ofHypoxylon. It is suggested that the coloured papillate species e.g.H. multiforme are more closely related toH. fragiforme and members of the Hypoxylon section than to non-coloured papillate species as indicated in the current classification of the genus.The relationships betweenHypoxylon, Daldinia andRosellinia are discussed.     

Effect of epiphytes on the extent of necrotic injuries of resistant and susceptible poplar clones infected with Dothichiza populea

Poplar cuttings of a resistant clone, Populus ‘Grandis’, and susceptible clones, Populus nigra ‘Italica’ and Populus ‘Robusta’, were infected with the pathogenic fungus Dothichiza populea alone, or with the pathogen and one of five strains of epiphytes antagonistic towards it (in vitro), isolated from poplar bark. The extent of injury was examined for 28 days after infection by determining the length of necrotic patches and their area as expressed in per cent of the total area of a cutting or the area of necrotic injuries caused by the pathogen alone.All the poplar cuttings of both the resistant and susceptible clones became diseased when infected with the pathogen alone. Surprisingly enough, however, the least affected clone was the susceptible P. ‘Robusta’, in which necrotic injuries covered 28% of the total area, as against 40% and 70% in the resistant P. ‘Grandis’ and the susceptible P. nigra ‘Italica’, respectively.When the cuttings were infected simultaneously with Dothichiza populea and its antagonistic epiphytes, the diseased area in the resistant clone diminished by as much as two-thirds, and in the susceptible P. nigra ‘Italica’, by one-third in comparison with the area affected by the pathogen alone. In turn, in the susceptible P. ‘Robusta’ the introduction of three out of five epiphytes stimulated the growth of the pathogenic fungus producing on average a double increase in the necrotic area. The differences in the response of the pathogen to the presence of epiphytes recorded in the susceptible clones indicate a marked influence of the plant on the nature of interactions between its epiphytic microflora and the pathogen.     

Field assessment of the risk posed by Diorhabda elongata, a biocontrol agent for control of saltcedar (Tamarix spp.), to a nontarget plant, Frankenia salina

The biological control program for saltcedar (Tamarix spp.) has led to open releases of a specialist beetle (Chrysomelidae: Diorhabda elongata) in several research locations, but the controversy over potential impacts to native, nontarget plants of the genus Frankenia remains unresolved. To assess the potential for nontarget impacts under field conditions, we installed cultivated Frankenia spp. (primarily two forms of Frankenia salina but also including Frankenia jamesii) at locations in Nevada and Wyoming where D. elongata densities and saltcedar defoliation were expected to be very high, so insects would be near starvation with high probability of attacking nontargets if these were suitable hosts. Subsequent insect abundance was high, and only minor impact (<4% foliar damage) was observed on both forms of F. salina under these ‘worst case’ conditions; there was no impact to F. jamesii. No oviposition nor larval development were observed on any plants, there was no dieback of damaged F. salina stems, and plants continued growing once insect populations subsided. These results under ‘natural’ field conditions contrast with caged host-range tests in which feeding, development and minor oviposition occurred on the nontarget plant. Other ecological factors, such as distance from target plants to natural Frankenia spp. populations, inhospitable conditions for agent survival in such sites, and intrinsic insect behavior that makes colonization and/or genetic adaptation highly unlikely, lead us to conclude that nontarget impacts following program implementation will be insignificant or absent. Host range testing of new agents, while necessary to ensure safety, must put greater attention on assessing the ecological context where agents will be establishing, and on balancing speculated risks against potential benefits of biological control.     

Molecular identification of endophytic fungi from medicinal plant <Emphasis Type="Italic">Huperzia serrata</Emphasis> based on rDNA ITS analysis

Fifty-two endophytic fungi strains with different colony morphologies were isolated from stems, leaves and roots of Huperzia serrata (Thunb. ex Murray) Trevis. collected from Bawangling Reserve of Hainan Province in southern China. They were identified mainly based on rDNA ITS sequences and phylogenetic analysis. The results showed that all strains belonged to four classes, i.e. Sordariomycetes (92.31%), Dothideomycetes (3.85%), Pezizomycetes (1.92%) and Agaricomycetes (1.92%). Forty-seven strains were identified at the genus level, including Glomerella (Colletotrichum), Hypocrea (Trichoderma), Pleurostoma, Chaetomium, Coniochaeta (Lecythophora), Daldinia, Xylaria, Hypoxylon, Nodulisporium, Cazia and Phellinus. As to the other five strains, three were identified at the order level and two at the family level, indicating that a great diversity of fungi taxa exists in H. serrata. Most isolated strains belonged to the genus of Glomerella (Colletotrichum) and Hypoxylon, twenty-one from Glomerella and its anamorph Colletotrichum (42.3% of total isolated strains) and ten from Hypoxylon (19.2% of total isolated strains). Pleurostoma, Chaetomium, Coniochaeta (Lecythophora), Daldinia, Xylaria, Hypoxylon, Nodulisporium, Cazia and Phellinus were reported as endophytic fungi isolated from H. serrata for the first time.     

Hypomiltin,a novel azaphilone from <Emphasis Type="Italic">Hypoxylon hypomiltum</Emphasis>, and chemotypes in <Emphasis Type="Italic">Hypoxylon</Emphasis> sect. <Emphasis Type="Italic">Hypoxylon</Emphasis> as inferred from analytical HPLC profiling

A novel azaphilone named hypomiltin was isolated by preparative reversed phase HPLC from the stromatal extract of the xylariaceous ascomycete Hypoxylon hypomiltum. Its chemical structure was determined by mass spectrometry and 2D nuclear magnetic resonance spectroscopy. Analytical HPLC profiling of stromatal crude extracts, using UV/visual (diode array) and mass spectrometric detection based on electrospray ionisation, revealed the presence of hypomiltin also in Hypoxylon intermedium, H. perforatum, H. trugodes, and Pulveria porrecta. In contrast, this compound was found neither in type material of H. hypomiltum var. lavandulocinereum nor in several further Hypoxylon species. Despite being chemically related to mitorubrin, hypomiltin never co-occurred with the latter compound and its derivatives. Characteristic secondary metabolite profiles of several further Hypoxylon species are correlated with the colours of their taxonomically significant KOH-extractable pigments. These species are divided into chemotypes, based on analytical HPLC data. The results point toward an extraordinary diversity of secondary metabolites in Hypoxylon. Dedicated to Timm Anke, Kaiserslautern, on the occasion of his 60th birthday     

New Mos1 mariner transposons suitable for the recovery of gene fusions in vivo and in vitro

The Drosophila Mos1 element can be mobilized in species ranging from prokaryotes to protozoans and vertebrates, and the purified transposase can be used for in vitro transposition assays. In this report we developed a ‘mini-Mos1’ element and describe a number of useful derivatives suitable for transposon mutagenesis in vivo or in vitro. Several of these allow the creation and/or selection of tripartite protein fusions to a green fluorescent protein–phleomycin resistance (GFP-PHLEO) reporter/selectable marker. Such X-GFP-PHLEO-X fusions have the advantage of retaining 5′ and 3′ regulatory information and N- and C-terminal protein targeting domains. A Mos1 derivative suitable for use in transposon-insertion mediated linker insertion (TIMLI) mutagenesis is described, and transposons bearing selectable markers suitable for use in the protozoan parasite Leishmania were made and tested. A novel ‘negative selection’ approach was developed which permits in vitro assays of transposons lacking bacterial selectable markers. Application of this assay to several Mos1 elements developed for use in insects suggests that the large mariner pM[cn] element used previously in vivo is poorly active in vitro, while the Mos1-Act-EGFP transposon is highly active.     

Pectenotoxin and okadaic acid-based toxin profiles in Dinophysis acuta and Dinophysis acuminata from New Zealand

The major pectenotoxin and okadaic acid group toxins in Dinophysis acuta and Dinophysis acuminata cell concentrates, collected from various locations around the coast of the South Island of New Zealand (NZ), were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). PTX2 and PTX11 were the major polyether toxins in all Dinophysis spp. cell concentrates. D. acuta contained PTX11 and PTX2 at concentrations of 4.7–64.6 and 32.5–107.5 pg per cell, respectively. The amounts of PTX11 and PTX2 in D. acuminata were much lower at 0.4–2.1 and 2.4–25.8 pg per cell, respectively. PTX seco acids comprised only 4% of the total PTX content of both D. acuta and D. acuminata. D. acuta contained low levels of OA (0.8–2.7 pg per cell) but specimens from the South Island west coast also contained up to 10 times higher levels of OA esters (7.0–10.2 pg per cell). Esterified forms of OA were not observed in D. acuta specimens from the Marlborough Sounds. D. acuta did not contain any DTX1 though all D. acuminata specimens contained DTX1 at levels of 0.1–2.4 pg per cell. DTX2 was not present in any New Zealand Dinophysis spp. specimens. Although the total toxin content varied spatially and temporally, the relative proportions of the various toxins in different specimens from the same location appeared to be relatively stable. The total PTX/total OA ratios in different isolates of D. acuta were very similar (mean±S.E.: 14.9±1.9), although the Marlborough Sounds D. acuminata isolates had a higher total PTX/total OA ratio (mean±S.E.: 22.7±2.4) than the Akaroa Harbour isolates (8.0). No evidence of azaspiracids were detected in these specimens. These results show that the LC–MS/MS monitoring of plankton for PTX group toxins (e.g. PTX2) and their derivatives (e.g. PTX2 seco acid) may provide a sensitive, semi-quantitative, indicator of the presence of more cryptic OA group toxins (e.g. OA esters).     

Isoenzyme diversity and cryptic speciation in Juniperus excelsa (Cupressaceae) complex in Iran

An isoenzyme analysis was performed on 109 individuals from 11 populations of Juniperus excelsa complex collected from Iran. A total of 61 allozymes from five enzyme systems were scored. The mean numbers of bands per presumed isoenzyme, ranged from 2.07 to 3.23. The value of Euclidean distance ranged from 1.920 to 4.411. The average value of Shannon diversity index (H′) estimated for each population ranged from 0.317 in the population sampled from ‘Fasa’ to 1.053 in the population of ‘Golestan’, and the average H′ value for each enzyme system ranged from 0.031 (SOD-3) to 1.670 (PPO-1). To reveal the relationships among these populations, a cluster analysis based on Euclidean distances was performed. The results of the analysis suggest recognizing three taxa in the complex in Iran: Juniperus polycarpos subsp. polycarpos, Juniperus polycarpos subsp. turcomanica, and one cryptic species not recorded from Iran until now. Moreover, the analysis presented, does not confirm the occurrence of J. excelsa in Iran.     

Importance of ascospore ornamentation in the taxonomy of Daldinia

Representative specimens of fifteen Daldinia spp. were studied for ultrastructural characteristics of their ascospores by scanning electron microscopy (SEM). The ornamentation of their outermost spore layers was found to be species-consistent, confirming the results of concurrent studies on the morphology of their teleomorphs and anamorphs, secondary metabolite profiles and PCR-based genetic fingerprints. Daldinia spp. may either show smooth or transversally striated ascospores. The spores of the species within the latter group are always ellipsoid-equilateral to ellipsoid-inequilateral with narrowly rounded ends. Smooth, broadly ellipsoid to cylindrical ascospores were observed in all species (D. caldariorum, D. fissa and D. loculata) that are known to produce their stromata on substrates damaged by fire. The ascospores of D. concentrica differed from those of D. childiae (i.e., the cosmopolitan taxon previously regarded as D. concentrica ss. auct.) and other Daldinia spp. in showing a very faint ornamentation, which only became visible at 10000× magnification by SEM. A specimen collected on the isle of Jersey (Channel Islands, UK) showed morphological similarities to the pantropical D. eschscholzii, but its ascospores appeared smooth by SEM, and it may therefore represent a previously undescribed species. Dedicated to Professor Yoshinori Asakawa, Tokushima, Japan, on the occasion of his 60^th birthday PH-R Life Science Center Natural Products     

Molecular phylogeny of Amoebozoa and the evolutionary significance of the unikont Phalansterium

The taxonomic position of the uniciliate, unicentriolar zooflagellate Phalansterium is problematic; its distinctive ultrastructure with a pericentriolar microtubular cone placed it in its own order and suggested phenotypic closeness to the eukaryote cenancestor. We sequenced the 18S rRNA of a unicellular Phalansterium. Phylogenetic analysis shows that it belongs to Amoebozoa, decisively rejecting a postulated relationship with the cercozoan Spongomonas; Phalansterium groups with Varipodida ord. nov. (Gephyramoeba/Filamoeba) or occasionally Centramoebida emend. (Acanthamoebidae/Balamuthiidae fam. nov.), centrosomes of the latter suggesting flagellate ancestors. We also studied Phalansterium solitarium cyst ultrastructure; unlike previously studied P. solitarium, this strain has pentagonally symmetric walls like P. consociatum. We also sequenced 18S rRNA genes of further isolates of Hyperamoeba, an aerobic unicentriolar amoeboflagellate with conical microtubular skeleton; both group strongly with myxogastrid Mycetozoa. However, the four Hyperamoeba strains do not group together, suggesting that Hyperamoeba are polyphyletic derivatives of myxogastrids that lost fruiting bodies independently. We revise amoebozoan higher-level classification into seven classes, establishing Stelamoebea cl. nov. for Protosteliida emend. plus Dictyosteliida (biciliate former ‘protostelids’ comprise Parastelida ord. nov. within Myxogastrea), and new subphylum Protamoebae to embrace Variosea cl. nov. (Centramoebida, Phalansteriida, Varipodida), Lobosea emend., Breviatea cl. nov. for ‘Mastigamoeba invertens’ and relatives, and Discosea cl. nov. comprising Glycostylida ord. nov. (vannellids, vexilliferids, paramoebids, Multicilia), Dermamoebida ord. nov. (Thecamoebidae) and Himatismenida. We argue that the ancestral amoebozoan was probably unikont and that the cenancestral eukaryote may have been also.     

Molecular Systematics of Middle American Cichlid Fishes and the Evolution of Trophic-Types in ‘Cichlasoma(Amphilophus)’ and ‘C.(Thorichthys)’

The majority of Middle American cichlids are placed in the informal assemblage ‘Cichlasoma.’ The group is divided into eight sections which appear to be based primarily on trophic morphology. Although several members of ‘Cichlasoma’ have been used in ecomorphological, behavioral, and biogeographic studies, no phylogenetic hypotheses for the group exist. In an attempt to develop a better understanding of the phylogenetic relationships of ‘cichlasomine’ cichlids, we examined the evolution of the trophic specialization, substratum-sifting, in two sections, ‘Cichlasoma(Thorichthys)’ and ‘C.(Amphilophus),’ to determine whether the trait reflects common ancestry. We sequenced the complete mitochondrial cytochromebgene for 19 cichlids representing six sections of ‘Cichlasoma,’ and representatives of other Neotropical Cichlidae. Additional cichlid, and noncichlid outgroup sequences were included for a total of 22 taxa. The molecular phylogeny supports the recognition of the section ‘C.(Thoricthys)’ as a natural group, and we place those cichlids in the genusThorichthys.The phylogeny also depicts ‘C.(Amphilophus)’ as paraphyletic, with substratum-sifters and generalized predators forming separate nonsister clades. We recommend that the substratum-sifting clade of the section ‘C.(Amphilophus)’ be placed in the resurrected genusAstatheros.The generalized predator clade of ‘C.(Amphilophus)’ contains only two species, ‘C. (A.) citrinellum’ and ‘C. (A.) labiatum,’ which we place in the genusAmphilophus.The phylogenetic hypotheses generated indicate that the substratum-sifting generaThorichthysandAmphilophusdo not share a common ancestor. Reconstruction of the evolution of substratum-sifting is equivocal, requiring either the independent evolution of the trait on two separate occasions or its presence in a more inclusive clade and subsequent loss in nonsubstratum sifting species.     

Identification of PaPKS1, a polyketide synthase involved in melanin formation and its use as a genetic tool in Podospora anserina

In the filamentous fungus Podospora anserina, many pigmentation mutations map to the median region of the complex locus ‘14’, called segment ‘29’. The data presented in this paper show that segment 29 corresponds to a gene encoding a polyketide synthase, designated PaPKS1, and identifies two mutations that completely or partially abolish the activity of the PaPKS1 polypeptide. We present evidence that the P. anserina green pigment is a (DHN)-melanin. Using the powerful genetic system of PaPKS1 cloning, we demonstrate that in P. anserina trans-duplicated sequences are subject to the RIP process as previously demonstrated for the cis-duplicated regions.     

DNA replication in macronuclei of Tetrahymena pyriformis GL

DNA replication in macronuclei of Tetrahymena pyriformis GL has been studied to discriminate between hypotheses developed for the interpretation of intraclonal differentiation in ciliated protozoa (the diploid subnuclear, and the ‘master’-‘slave’ hypotheses). Tetrahymena cells were grown in a heavy ¹⁵N-³H medium and then transferred to a light ¹⁴N-¹⁴C medium. DNA was isolated after various periods following this transfer and studied in equilibrium CsCl density gradient centrifugation. Time-related changes in the DNA buoyant density pattern were investigated. The data obtained are interpreted to mean that all DNA in macronuclei of asynchronously growing Tetrahymena at exponential phase replicates semiconservatively once in a cell cycle. These data are in good agreement with the findings of Andersen & Zeuthen obtained on synchronous Tetrahymena cultures in the presence of BUdR.These results are not consistent with the ‘master’-‘slave’ hypothesis. The diploid subnuclear hypothesis is not in accord with other experimental evidence. An alternative hypothesis has been proposed concerning the nature of the macronuclear units and the process of determination. The two main points of this hypothesis are: (a) macronuclear units are diploid genome fragments (‘nucleosomes’); (b) determination is a process of haploidization by ‘allelic splitting’ at a definite macronuclear fission. Consistency with experimental data is discussed and some predictions of the hypothesis are given.     

Phenology and floral visitors of two sympatric Heliconia species in the Brazilian Atlantic forest

The main goal of the present study was to test if two sympatric species of Heliconia, Heliconia spathocircinata and Heliconia laneana var. flava, differ in their reproductive seasons and guilds of flower visitors. Twenty-four sampling sessions were conducted monthly between February 2001 and January 2003. Individuals of H. spathocircinata and of H. laneana var. flava were tagged within a transect of 4000×4 m. Reproductive phenophases (flowering, unripe, and ripe fruits) were recorded, and circular statistics were used to test for the regularity and frequency of phenophases. Flower visitors were observed during two flowering periods for each species. Both species had well-defined and synchronous reproductive periods, with a small overlap, suggesting a sequential flowering model. Biotic and abiotic factors seem to affect phenophases of both species. However, herbarium data suggest that the flowering period in H. spathocircinata varies less than in H. laneana, despite its wider geographic distribution. The hummingbird Phaethornis idaliae was the main floral visitor of both Heliconia species studied. H. laneana was also legitimately visited by another hummingbird species, Glaucis hirsuta, and by Lepidoptera. Some Lepidoptera, particularly the bee Trigona spinipes, were illegitimate visitors (‘nectar robbers’). We suggest that the flower of H. spathocircinata allowed a wider array of visitors, because it is bigger.     

In vitro cytogenetic effects of Andrographis paniculata (kalmegh) on arsenic

In vitro effects of arsenic in human peripheral lymphocytes (HPL) at three different doses – 3.6×10⁻⁴, 1.4×10⁻³ and 0.72×10⁻³ μM for 24 h before harvesting on sister chromatid exchanges (SCE), Cell cycle proliferative index/replicative index (CCPI/RI), %M₁, %M₂ and %M₃, population doubling time (PDT) and average generation time (AGT) were examined. Andrographis paniculata (commonly referred to as ‘kalmegh’) has been used for centuries in traditional Indian and Chinese herbal medicine as a safe, natural folk remedy for assorted health concerns. In the present study, kalmegh (0.01 μg/7 ml culture media) was used along with the highest dose of arsenic; the results showed that arsenic induced increase in these genotoxic endpoints were fairly diminished by kalmegh. In addition, mutagenic in vitro effect of ethyl methanesulphonate (EMS) was used as a positive control in this study. It is thus concluded from this study that Andrographis has a protective role in arsenic toxicity.