Multiple, novel biologically active endophytic actinomycetes isolated from upper Amazonian rainforests.

Microbial biodiversity provides an increasingly important source of medically and industrially useful compounds. We have isolated 14 actinomycete species from a collection of approximately 300 plant stem samples from the upper Amazonian rainforest in Peru. All of the cultured isolates produce substances with inhibitory activity directed at a range of potential fungal and bacterial pathogens. For some organisms, this activity is very broad in spectrum while other organisms show specific activity against a limited number of organisms. Two of these organisms preferentially inhibit bacterial test organisms over eukaryotic organisms. rDNA sequence analysis indicates that these organisms are not equivalent to any other cultured deposits in GenBank. Our results provide evidence of the untapped biodiversity in the form of biologically active microbes present within the tissues of higher plants.

Synergism among volatile organic compounds resulting in increased antibiosis in Oidium sp.

Oidium sp. has been recovered as an endophyte in Terminalia catappa (tropical chestnut) in Costa Rica. The volatile organic compounds (VOCs) of this organism uniquely and primarily consist of esters of propanoic acid, 2-methyl-, butanoic acid, 2-methyl-, and butanoic acid, 3-methyl-. The VOCs of Oidium sp. are slightly inhibitory to many plant pathogenic fungi. Previous work on the VOCs of Muscodor albus demonstrated that besides esters of small organic acids, a small organic acid and a naphthalene derivative were needed to obtain maximum antibiotic activity. Thus, the addition of exogenous volatile compounds such as isobutyric acid and naphthalene, 1,1'-oxybis caused a dramatic synergistic increase in the antibiotic activity of the VOCs of Oidium sp. against Pythium ultimum. In fact, at elevated concentrations, there was not only 100% inhibition of P. ultimum but killing as well. In addition, a coculture of Muscodor vitigenus (making only naphthalene) and Oidium sp. plus isobutyric acid produced an additive antibiosis effect against P. ultimum. The biological implications of multiple volatile compounds acting to bring about antibiosis in nature are discussed.

Bioactive endophytic streptomycetes from the Malay Peninsula.

Three novel endophytic streptomycetes have been isolated and characterized from plants with ethnobotanical uses on the Malay Peninsula including: Thottea grandiflora (family -Aristolochiaceae), Polyalthia spp. (family -Annonaceae), and Mapania sp. (family -Cyperaceae). Each isolate, as studied by scanning electron microscopy, has small hyphae, and produces typical barrel-shaped spores arising by hyphal fragmentation. Interestingly, although none has any detectable antibacterial killing properties, each has demonstrable killing activity against one or more pathogenic fungi including organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis and Rhizoctonia solani. Molecular biological studies on the rRNA gene sequence of each isolate revealed that it is distinct from all other genetic accessions of streptomyectes in GenBank, and each bears some genetic similarity to other streptomycetes. The bioactivity of each microbe was extractable in various organic solvents.

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.

Scanning electron microscopy of some endophytic streptomycetes in snakevine--Kennedia nigricans.

Soils of all types and locations have generally served as the major sources of streptomycetous bacteria. These organisms are the source of nearly 80% of the world's antibiotics. Now, it is realized that Streptomyces spp. (within the group of prokaryotic filamentous bacteria known as actinomycetes) can exist as endophytes within the interstices of some higher plants. While it is sometimes possible to isolate one or two different streptomycetes from certain plants, most plants are free of these organisms. However, the snakevine (Kennedia nigricans) of the Northern Territory of Australia has yielded at least 39 different endophytic actinomycetes (95% of them being Streptomyces spp.) Most of these isolates possessed no detectable antibiotic properties, while at least seven had antibacterial and antifungal activities. Examination of eight selected cultures by scanning electron microscopy (SEM) as well as environmental scanning electron microcopy (FEI ESEM FEG) (FEI Company, Hillsobro, Ore., USA) revealed unusual patterns, structures, and features of the spores and hyphae of these microorganisms. For instance, as revealed by ESEM FEG for the first time, it has become obvious that extremely fine hair-like structures (average 25-49 nm with gold-coated specimens) exist on the spores and hyphae of some endophytic streptomycetes. The biological purpose of these hair-like protrusions is unknown. Both SEM and ESEM FEG can be effectively used as tools in identification and elucidation of the biology of these organisms. In addition, unusual colony morphology, observed with the unaided eye can very easily be used to distinguish some of these isolates since characteristic donut and pseudo-horn shaped colonies appeared in culture.

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.

Biologically active endophytic streptomycetes from Nothofagus spp. and other plants in Patagonia.

Endophytic streptomycetes have been isolated and characterized from several species of Nothofagus and other plants growing in the southern reaches of Patagonia. No endophytic streptomycete was obtained from any plant species studied in Northern Patagonia. However, from Southern Patagonia, biologically active Streptomyces spp. from several plant species were isolated. Each isolate, as studied by scanning electron microscopy (SEM), has small hyphae, some produce typical barrel-shaped spores in culture and each has some unique hyphal surface structures. Interestingly, although none has any detectable antibacterial killing properties, each has demonstrable killing activity against one or more pathogenic fungi including representative plant pathogenic organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis, and Rhizoctonia solani. The 16S rDNA sequences of the isolates were distinct from all other genetic accessions of Streptomyces in GenBank. However, isolate C-2 from Chiliotrichum diffusum (Compositae) is identical, in all respects, to isolate C-4 obtained from Misodendrum punctulatum (Loranthaceae). These results confirm that endophytic streptomycetes represent a novel source of biologically active microorganisms.

Development and nature of the partition layer in Tilletia caries teliospore walls.

Developing Tilletia caries teliospores were studied with thin sectioning procedures. After the W1 and W2 spore walls are formed, lamellar material begins to form adjacent to the W2 wall layer. The patches of lamellar material become continuous, and additional layers are added. After the W3 wall starts to form, the lamellar material is difficult to see without special staining. The lamellar material makes it difficult to get resins to penetrate the partition layer of teliospore walls.

Ultrastructure of lipid bodies in Tilletia caries teliospores.

The ultrastructure of lipid bodies within developing, dormant, and germinating Tilletia caries (DC). Tul. (race T-16) teliospores was studied by freeze-etching and thin-sectioning techniques. When teliospores were prefixed in sodium cacodylate-buffered glutaraldehyde-acrolein for 24 h before further processing, most of the lipid bodies appeared to have a uniformly osmiophilic matrix. Some of these lipid bodies were surrounded by thin electron-dense lines that appeared to be half-unit membranes. Occasionally this membrane seemed to be absent, allowing for a direct interface between lipid and cytosol. Irregular electron-dense patterns were occasionally observed in lipid bodies of developing, dormant, and germinating teliospores. A lamellar substructure with 6- to 10-nm center-to-center spacing was visible in the electron-dense patterns at high magnifications. Irregular fracture patterns were visible in freeze-etch replicas.

Evidence for the presence of the toxin-binding protein on the plasma membrane of sugarcane cells.

Helminthosporoside is a host-specific toxin produced by Helminthosporium sacchari, both in culture and in infected sugarcane tissue. The susceptibility of any given clone of sugarcane to disease is invariably associated with the presence of a toxin-binding protein. This report presents evidence for the presence of the toxin-binding protein on the plasma membrane of the plant cell. This evidence includes, among others: (1) The protection of susceptible plant tissues by prior treatment with antiserum to the binding protein, (2) the successful pyridoxylation and reduction of the binding protein in vivo followed by its isolation, (3) the reaction of sugarcane cells and free protoplasts with [(3)H]antiserum prepared against the binding protein, and (4) the agglutination of sugarcane protoplast preparations with antiserum to the binding protein.

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.

New endophytic isolates of Muscodor albus, a volatile-antibiotic-producing fungus.

Muscodor albus, an endophytic fungus originally isolated from Cinnamomum zeylanicum, produces a mixture of volatile organic compounds (VOCs) in culture and its spectrum of antimicrobial activity is broad. Using the original isolate of M. albus as a selection tool, it has been possible to find other culturally and biochemically unique wild-type isolates of this organism existing as endophytes in a variety of other plant species, including Grevillea pterifolia (fern-leafed grevillea), Kennedia nigriscans (snake vine) and Terminalia prostrata (nanka bakarra) growing in the northern reaches of the Northern Territory of Australia. Interestingly, none of the new isolates had a culture morphology that was identical to the original isolate, nevertheless each possessed hyphal characteristics that resembled that isolate. Furthermore, their ITS-5.8S rDNA sequences were 96-99 % identical to that of M. albus and the isolates were considered M. albus on the basis of the DNA sequence data. However, the VOCs produced by these new isolates greatly differed in quality from the original strain by virtue of the production of naphthalene, naphthalene, 1,1'-oxybis-, and one or more other compounds. In bioassays with a range of test micro-organisms, including fungi and bacteria, each isolate possessed biological activity but the range of activity was great. Artificial mixtures of some of the VOCs mimicked the effects of the VOCs of the fungus. The value of these observations to the biology and practical uses of M. albus in agriculture and other applications is discussed.

Surface rodlets of Tilletia indica teliospores.

Tilletia indica teliospores were studied by use of thin sections and freeze-etch replicas. Surfaces of these spores have rodlet patterns which differ from those previously reported for spores of other fungi. The rodlets on T. indica teliospores average 240 nm in length and are not grouped into fascicles.

Fixation of dormant Tilletia teliospores for thin sectioning.

Dormant Tilletia caries teliospores in fixative solution or distilled water were frozen onto specimen chucks of an FTS Sorvall-Christensen frozen thin sectioner and cut or fractured at various temperatures (-20 C to -75 C) and thickness settings (10, 15, 20 and 25 mum). Cytoplasm of dormant spores was well preserved and organelles were found to differ from those of germinated spores in morphology. This procedure makes it possible to fix adequately dormant spores and thus compare the ultrastructure and histochemistry of dormant spores with those of germinated spores.