Comparison of Muscodor albus Volatiles with a Biorational Mixture for Control of Seedling Diseases of Sugar Beet and Root-Knot Nematode on Tomato.

A biorational synthetic mixture of organic components mimicking key antimicrobial gases produced by Muscodor albus was equivalent to the use of live M. albus for control of seedling diseases of sugar beet (Beta vulgaris) caused by Pythium ultimum, Rhizoctonia solani AG 2-2, and Aphanomyces cochlioides. The biorational mixture provided better control than the live M. albus formulation for control of root-knot nematode, Meloidogyne incognita, on tomato (Lycopersicon esculentum). The biorational mixture provided control of damping-off equal to a starch-based formulation of the live fungus for all three sugar beet pathogens, and significantly reduced the number of root-knot galls on tomato roots compared with a barley-based formulation. Rate studies with the biorational mixture showed that 2 and 0.75 µl/cm3 of soil were required to provide optimal control of Rhizoctonia and Pythium damping-off of sugar beet, respectively. Five microliters of biorational mixture per milliliter of water was required for 100% mortality in 24 h for Meloidogyne incognita in in vitro studies. In in vivo studies, 1.67 µl of the biorational mixture/cm3 of sand resulted in fewer root-knot galls than a Muscodor albus infested ground barley formulation applied at 5 g/liter of sand.

Mycofumigation with Muscodor albus and Muscodor roseus for Control of Seedling Diseases of Sugar Beet and Verticillium Wilt of Eggplant.

Mycofumigation is the use of antimicrobial volatiles produced by fungi such as Muscodor albusitalic and M. roseus for the control of other organisms. Sugar beet (Beta vulgaris L.) stand establishment was increased and disease severity decreased by mycofumigation with M. roseus and M. albus in autoclaved soil infested with Rhizoctonia solani, Pythium ultimum, or Aphanomyces cochlioides. Eggplant seedlings (Solanum melongena L.) transplanted into autoclaved soil infested with Verticillium dahliae and mycofumigated with M. albus and M. roseus had significantly less disease (P < 0.05) after 4 and 5 weeks compared with nonmycofumigated Verticillium-infested soil. The effect of formulation on efficacy of mycofumigation with M. roseus was tested using potato dextrose agar strips, alginate capsules, ground barley, pesta granules, and stabileze granules. The stabileze and ground barley formulations of M. roseus resulted in the best control of P. ultimum damping-off. The best control of A. cochlioides damping-off was with the stabileze formulation, and the stabileze, ground barley, and agar strip formulations provided similar control of R. solani damping-off. In soil infested with P. ultimum, mycofumigation with M. albus stabileze formulation resulted in stand establishment similar to that in the autoclaved soil. Mycofumigation was ineffective in controlling Fusarium wilt of sugar beet. Neither M. albus nor M. roseus affected sugar beet or eggplant growth or appearance except in the stabileze formulation, where stunting was noticed. Mycofumigation with M. albus and M. roseus shows promise for control of soilborne diseases caused by P. ultimum, A. cochlioides, R. solani, and V. dahliae.

Volatile antimicrobials from Muscodor albus, a novel endophytic fungus.

Muscodor albus is a recently described endophytic fungus obtained from small limbs of Cinnamomum zeylanicum (cinnamon tree). This xylariaceaous fungus effectively inhibits and kills certain other fungi, and bacteria, by virtue of a mixture of volatile compounds that it produces. The majority of these compounds were identified by gas chromatography/mass spectrometry and then made into an artificial mixture that mimicked the antibiotic effects of the mixture of volatile compounds given off by the fungus. Each of the five classes of volatile compounds produced by the fungus (alcohols, esters, ketones, acids and lipids) had some inhibitory effect against the test fungi and bacteria, but none was lethal. However, collectively they acted synergistically to kill a broad range of plant- and human-pathogenic fungi and bacteria. The most effective class of inhibitory compounds was the esters, of which 1-butanol, 3-methyl-, acetate was the most active biologically. This report describes the ecological implications and potential practical benefits of the 'mycofumigation' effects of M. albus.

Characterization of stereochemistry and molecular conformation using solid-state NMR tensors.

A solid-state NMR technique is described for establishing stereochemistry using the natural product terrein as a model compound. This method involves comparison of experimental (13)C tensor principal values with ab initio computed values for all possible computer-generated stereoisomers. In terrein the relative stereochemistry is confirmed by NMR to be 2R*,3S with high statistical probability (>99.5%). The proposed approach also simultaneously verifies the molecular conformation of the two hydroxy groups in terrein established by X-ray data. It is sufficient to use only shift tensor values at carbons 2 and 3, the stereocenters, to characterize both the stereochemistry and molecular conformations. The solid-state NMR method appears to be especially useful for determining relative stereochemistry of compounds or their derivatives that are difficult to crystallize.

Two telomerase reverse transcriptases (TERTs) expressed in Candida albicans.

The human pathogenic yeast Candida albicans contains two telomerase reverse transcriptase (TERT) genes. CaTERT1 and CaTERT2 appear either to be two alleles of the same gene or two entirely different genes that encode 867-residue proteins that differ by five amino acids. Both TERTs have a calculated pI of 9.5 and a M(r) of 100.9 kDa and are the smallest TERTs identified to date. Both genes appear to be expressed. Based on sequence similarity between CaTERT1 and the Saccharomyces cerevisiae orthologue Est2p, we suggest a revised alignment for motif E of Est2p. The identification of these TERT genes provides the first opportunity to study telomerase in an important human pathogen.

Evidence for paclitaxel from three new endophytic fungi of Himalayan yew of Nepal.

Taxus wallichiana, collected from the Himalayan region of Nepal, yielded, among others, three different endophytic fungi including Sporormia minima, Trichothecium sp., and an unidentified dimorphic fungus. Each was shown to produce paclitaxel in a culture medium. Each of these fungi represents a new report as a paclitaxel producer. The production of paclitaxel was confirmed by an immunoassay that utilized monoclonal antibodies, TLC chromatography as well as high performance liquid chromatography combined with on-line mass spectrometry.

Jesterone and hydroxy-jesterone antioomycete cyclohexenone epoxides from the endophytic fungus Pestalotiopsis jesteri.

Jesterone and hydroxy-jesterone are novel highly functionalized cyclohexenone epoxides isolated from a newly described endophytic fungal species--Pestalotiopsis jesteri. They were purified from cultures of the fungus by bioassay guided fractionation using Pythium ultimum as the indicator organism. Jesterone, in particular, displays selective antimycotic activity against the oomycetous fungi which are some of the most plant pathogenic of all disease causing fungi. The possible importance of these cyclohexenones to the biology of the endophytic fungus-host plant relationship is also discussed.

Ambuic acid, a highly functionalized cyclohexenone with antifungal activity from Pestalotiopsis spp. and Monochaetia sp.

Ambuic acid, a highly functionalized cyclohexenone, was isolated and characterized from Pestalotiopsis spp. and Monochaetia sp. these being biologically related endophytic fungi associated with many tropical plant species. This compound was found in representative isolates of these fungal species obtained from rainforest plants located on several continents. The relevance of ambuic acid to the biology of the association of these fungi to their host plants is also discussed.

Seimatoantlerium tepuiense gen. nov., a unique epiphytic fungus producing taxol from the Venezuelan Guyana.

Seimatoantlerium gen. nov., type species, S. tepuiense sp. nov. is proposed for an acervular fungus producing 4-septate, holoblastic conidia with 6-8 unbranched, apical appendages that dehisce as an appendage apparatus and also commonly possessing one or two exogenous basal appendages as well as a pedicel. It is compared with Seimatosporium, Seimatosporiopsis, and other genera. It is epiphytic on Maguireothamnus speciosus, a rubiaceous plant endemic to the tepuis of southeastern Venezuela. It produces the anti-oomycetous anticancer compound, taxol, as shown by immunological and spectroscopic methods. Taxol production is discussed relative to the ability of this fungus to exist in an extremely moist ecosystem, as well as to its relationship to other plant associated fungi.

Seimatoantlerium tepuiense gen. nov., a unique epiphytic fungus promoting taxol from the Venezuelan Guyana

Seimatoantlerium gen. nov., type species, S.tepuiense sp. nov. is proposed for an acervular fungus producing 4-septate, holobastic conidia with 6-8 unbranched, apical appendages that dehisce as an appendage apparatus and also commonly possessing one or two exogenous basal appendages as well as a pedicel. It is compared with Seimatosporium, Seimatosporiopsis, and other genera. It is epiphytic on Maguireothamnus speciosus, a rubiaceous plant endemic to the tepuis of southeastern Venezuela. It produces the anti-oomycetous anticancer compound, taxol, as shown by immunological and spectroscopic methods. Taxol production is discussed relative to the ability of this fungus to exist in an extremely moist ecosystem, as well as to its relationship to other plant associated fungi. (AU)

In vivo addition of telomeric repeats to foreign DNA generates extrachromosomal DNAs in the taxol-producing fungus Pestalotiopsis microspora.

Transformation of the taxol-producing filamentous fungus Pestalotiopsis microspora with a plasmid containing the bacterial hygromycin resistance gene fused to Aspergillus regulatory sequences resulted in the in vivo formation of extrachromosomal DNAs with telomeric repeats in the majority of transformants. Repeats of the telomeric sequence 5'-TTAGGG-3' were appended to nontelomeric transforming DNA termini. No fungal sequences other than telomeric repeats were detected in extrachromosomal DNAs. Transformants contained three to six different sizes or conformational forms of extrachromosomal DNAs. The DNAs showed no change in size or internal structure during 6 months of growth with selection, but were lost after 20 days of growth without selection. Transformation of wild-type P. microspora with a PCR-amplified extrachromosomal DNA having terminal telomeric repeats produced up to 50-fold more transformants than the original transformation vector. The addition of telomeric repeats to foreign DNA is unusual among fungi and may have important adaptive or developmental implications.

Ecomycins, unique antimycotics from Pseudomonas viridiflava.

A novel family of peptide antimycotics, termed ecomycins, is described from Pseudomonas viridiflava, a plant-associated bacterium. Ecomycins B and C have molecular masses of 1153 and 1181. They contain equimolar amounts of a beta hydroxyaspartic acid, homoserine, threonine, serine, alanine, glycine and one unknown amino acid. Fatty acids were detectable after hydrolysis, methylation and gas chromatography and mass spectroscopy. The ecomycins have significant bioactivities against a wide range of human and plant pathogenic fungi. The minimum inhibitory concentration values for ecomycin B were 4.0 micrograms ml-1 against Cryptococcus neoformans and 31 micrograms ml-1 against Candida albicans. Pseudomonas viridiflava also produces what appears to be syringotoxin, an antifungal lipopeptide previously described from Ps. syringae.

Glucosylation of the peptide leucinostatin A, produced by an endophytic fungus of European yew, may protect the host from leucinostatin toxicity.

BACKGROUND: Yew species (Taxus spp.) throughout the world are hosts to hundreds, or perhaps thousands, of endophytic organisms. Most commonly, these organisms are fungi, living in a commensal or a symbiotic relationship with their host plant, so the plants exhibit little or no outward evidence that they are supporting these microorganisms. Little is known about any of the biochemical mechanisms that mediate the interactions between the yew host and its associated microbes. We feel that such information may not only contribute to our understanding of endophyte-tree biology, but also may provide novel pharmaceutical leads, because some of the compounds produced by these endophytes have demonstrated pharmacological activities. RESULTS: Acremonium sp. was isolated as an endophytic fungus of the European yew, Taxus baccata. Entry of Acremonium sp. into the plant may proceed via invasion of natural openings such as stomata. The relationship between Acremonium sp. and T. baccata may be a symbiotic one, because no symptoms are seen when Taxus media p.v. Hicksii is inoculated with this fungus. In culture, the fungus makes leucinostatin A, a peptide with phytotoxic, anticancer and antifungal properties. Although this peptide causes necrotic symptoms in many non-host plants and other cell types, it causes no visible symptoms in the host plant. T. baccata and several other plants have a UDP glucose; leucinostatin A glucosyl transferase that catalyzes the production of leucinostatin A beta di-O-glucoside from leucinostatin A. This glucoside, also made by the fungus, has a lower bioactivity against plants, fungi and a breast cancer cell line, BT-20, than leucinostatin A. CONCLUSIONS: Leucinostatin A may be one of several potentially toxic peptides produced by Acremonium sp. that contribute to the defense of the host, thereby preserving the fungus' own biological niche. The host plant is relatively immune to leucinostatin A because it has an enzyme which transfers two glycosyl residues to leucinostatin A, markedly reducing the peptide's bioactivity. Our results suggest that glucosylation reactions may play a more general role in plant defenses, especially against toxin-mediated disease development.

A scanning electron microscopy study of Taxus leaves as related to taxonomy.

Scanning electron microscopy, when applied to the surfaces of the needles of Taxus spp. (yew) revealed features that appear useful in the taxonomy of this tree species which yields the important anticancer drug, taxol. For instance, all of the four North American species have 3-5 rows of stomata on one-half of the abaxial leaf surface, whereas all of the others, including those from Europe and Asia, have 7-10 rows of stomata. The appearance of individual or fused papilliform epidermal cells and their arrangement on the leaf surface also is a feature that varies between species. Patterns of wax formation appeared on all species of yew examined but none could characteristically be assigned to a given species. Wax pattern variation was dependent upon age, environment, and probably to some extent, species differences. This study may provide some additional useful and reliable indicators in Taxus taxonomy.

Novel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins.

The covalent structure and most of the stereochemistry of the pseudomycins, bioactive metabolites of a transposon-generated mutant of a Pseudomonas syringae wild-type strain proposed for the biological control of Dutch elm disease, have been determined. While two pseudomycins are identical to the known syringopeptins 25-A and 25-B, pseudomycins A, B, C, C' are new lipodepsinonapeptides. For all of these the peptide moiety corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH) -L-Thr (4-Cl) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N-terminal Ser. This is in turn N-acylated by 3,4-dihydroxytetradecanoate in pseudomycin A, by 3-hydroxytetradecanoate in pseudomycin B, by 3,4-dihydroxyhexadecanoate in pseudomycin C, and by 3-hydroxyhexadecanoate in pseudomycin C'. Some preliminary data on the biological activity of pseudomycin A are reported.

Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.).

Maculosin (the diketopiperazine, cyclo (L-Pro-L-Tyr)) is a host specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa L.). Receptors for this phytotoxin have been isolated from spotted knapweed. Knapweed leaves possess most of the maculosin-binding activity in the cytosolic fraction. However, activity was also observed in the whole membrane fraction of the leaf. The binding component of the cytosolic fraction was identified as a protein(s) because of its heat-lability and sensitivity to proteases. A 16-fold purification of a toxin-binding protein was carried out by ammonium sulfate fractionation, and Sephadex G-200, and maculosin-affinity column chromatography. The affinity column was prepared with epoxy activated Sepharose 6B to which the phenolic group of maculosin was attached. The receptor was estimated to contain more than one binding protein by native and SDS-PAGE. At least one of the maculosin-binding proteins was identified as ribulose-1,5-biphosphate carboxylase (RuBPcase).

Maculosin, a host-specific phytotoxin for spotted knapweed from Alternaria alternata.

Several diketopiperazines have been isolated from liquid cultures of Alternaria alternata, the causal agent of black leaf blight of spotted knapweed, Centaurea maculosa Lam. One of these compounds, maculosin [the diketopiperazine cyclo(-L-Pro-L-Tyr-)], was active in the nicked-leaf bioassay at 10(-5) M; synthetic maculosin possessed chemical and biological activities identical to those of the natural product. Other diketopiperazines isolated from the fungus possessed either less activity or none at all. In tests against 19 plant species, maculosin was phytotoxic only to spotted knapweed. Thus maculosin is a host-specific phytotoxin from a weed pathogen.

Transposon mutagenesis and tagging of fluorescent Pseudomonas: Antimycotic production is necessary for control of Dutch elm disease.

Antimycotic-producing strains of Pseudomonas syringae are being tested as Dutch elm disease control agents. We examined the role of antimycotic production in disease control. Transposon Tn903 was used to mutagenize the antimycotic-producing strain MSU174. Eighty-one mutants that did not inhibit fungal growth were identified among 15,000 Tn903-containing derivatives. Linkages between Tn903 insertions and defects in antimycotic metabolism were established. Three Tn903-containing strains (two antimycotic producers and one nonproducer) were individually introduced into American elm seedlings. The seedlings were subsequently challenged with Ceratocystis ulmi, the causal agent of Dutch elm disease. Protection of the elms was observed with the two antimycotic-producing strains but not with the nonproducing strain. The introduced strains could be readily recovered from the seedlings after two growing seasons. They were unequivocally identified by the Tn903 insertions they contain.