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.

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.

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.

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.

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.

Induction of the sexual stage of Pestalotiopsis microspora, a taxol-producing fungus.

Pestalotiopsis microspora, isolate NE-32, is an endophyte of the Himalayan yew (Taxus wallichiana) that produces taxol, an important chemotherapeutic drug used in the treatment of breast and ovarian cancers. Conditions were determined to induce the perfect stage (teleomorph) of this organism in the laboratory as a critical first step to study inheritance of taxol biosynthetic genes. The perfect stage of Pestalotiopsis microspora NE-32 forms in a period of 3-6 weeks on water agarose with dried yew needles at 16-20 degrees C with 12 h of light per day. Morphological analysis of the teleomorph and sequencing of the 18S rDNA indicates that Pestalosphaeria hansenii is the perfect stage of Pestalotiopsis microspora. Only certain plants (e.g. yews, some pines, pecan, oat and some barley cultivars) allow the production of perithecia. Exhaustive methylene chloride extraction of yew (Taxus cuspidata) needles removes their capacity to induce production of perithecia. The methylene chloride extract is able to induce formation of perithecia by strain NE-32 in a bioassay system utilizing the sterilized sheaths of the Cholla cactus (Opuntia bigelovii) spine, indicating that a chemical compound(s) in yew stimulates the formation of the perfect stage. This hydrophobic plant compound(s) has been designated the perithecial-stimulating factor (PSF). The data suggest that plant products may play a role in regulating the biology of endophytic microbes.

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)

Cryptocandin, a potent antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina.

A unique lipopeptide antimycotic, termed cryptocandin, is described from Cryptosporiopsis cf. quercina, an endophytic fungus. Cryptocandin, with a molecular mass of 1079 Da, contains equimolar amounts of 3,4-dihydroxyhomotyrosine, 4-hydroxyproline, threonine, glutamine, 3-hydroxy-4-hydroxymethylproline, 4,5-dihydroxyornithine and palmitic acid. Cryptocandin is chemically related to well-known antimycotics, the echinocandins and pneumocandins, which are produced by such fungi as Zalerion arboricola, Pezicula spp. and Aspergillus spp. Cryptocandin has minimal inhibitory concentration values of 0.03-0.07 microgram ml-1 against isolates of Candida albicans, Trichophyton mentagrophytes and Trichophyton rubrum. Cryptocandin is also active against a number of plant-pathogenic fungi including Sclerotinia sclerotiorum and Botrytis cinerea.

Oocydin A, a chlorinated macrocyclic lactone with potent anti-oomycete activity from Serratia marcescens.

A unique chlorinated macrocyclic lactone, termed oocydin A, was isolated from a strain of Serratia marcescens growing as an epiphyte on Rhyncholacis pedicillata, an aquatic plant native to the Carrao river of the Venezuelan-Guyanan region of South America. The lactone has a molecular mass of 470 Da, and contains one atom of chlorine, a carboxyl group and a tetrahydrofuran ring internal to a larger macrocyclic ring. MICs of approximately 0.03 microg ml(-1) were noted for oocydin A against such phytopathogenic oomycetes as Pythium ultimum, Phytophthora parasitica, Phytophthora cinnamomi and Phytophthora citrophora. With regard to the true fungi, oocydin A had either minimal or no effect against certain Fungi Imperfecti (including several pathogens of humans), two ascomycetes and a basidiomycete. Oocydin A may have potential as an antimycotic in agricultural applications and especially for crop protection.

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.

Endophytic taxol-producing fungi from bald cypress, Taxodium distichum.

Pestalotiopsis microspora occurs as a range of strains in bald cypress, Taxodium distichum. The organisms live as endophytes in the bark, phloem and xylem, and isolates show differences in cultural and microscopic characteristics on common laboratory media. Many of these fungi make taxol as determined by the reactivity of partially purified culture extracts with specific monoclonal antibodies against taxol. In the case of one strain of P. microspora (CP-4), taxol was isolated from culture medium and was shown to be identical to authentic taxol by chromatographic and spectroscopic means.

Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana.

Pestalotiopsis microspora was isolated from the inner bark of a small limb of Himalayan yew, Taxus wallachiana, and was shown to produce taxol in mycelial culture. Taxol was identified by spectroscopic and chromatographic comparisons with authentic taxol. Optimal taxol production occurred after 2-3 weeks in still culture at 23 degrees C. [14C]Acetate and [14C]phenylalanine served as precursors for fungal [14C]taxol. These observations on P. microspora are discussed in relation to the biological importance of taxol production by fungi in general.

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.

Human hair morphology: a scanning electron microscopy study on a male Caucasoid and a computerized classification of regional differences.

The present study was performed to provide a better understanding of the morphological variations of mammalian hair. Terminal hair samples were obtained from different regions of the body of the same Caucasian male. All hair samples were either cleaned or treated before being examined with scanning electron microscopy. As human scalp hair grew it appeared small like lanugo hair, but the increase in diameter appeared to have been relatively rapid. As hair increased in diameter the appearance of the scales changed. Neck hair was slightly smaller in diameter than scalp hair, and axillary hair was slightly smaller in diameter than neck hair. Nostril hair was larger than scalp or axillary hair. Eyelash hair was much smaller and much shorter than eyebrow hair. Neck hair, forearm hair, and shin hair were smaller than hair from most other regions of the body. Chest hair was similar in size to scalp hair, and pubic and sideburn hair were larger than scalp hair. A morphological feature called "steak-boning" was more characteristically present in whiskers of Caucasoids than Orientals or Blacks. "Steak-boning" occurred most frequently in hair of the mustache, followed by that of the chin, sideburn, cheek and under the chin. Cut surfaces of whiskers were different for electric as compared with straightedge razors. Hair morphology varied relative to the body region. Computer analysis of resin-embedded hair made it possible to classify arm, mustache, cheek, chin, head, shin, and pubic hair, and to quantify cross-sectioned differences.

Role of pit membranes in macromolecule-induced wilt of plants.

Macromolecules present in low concentrations in xylem fluid of Medicago sativa L. var DuPuits will increase the resistance to xylem liquid flow. This increase in resistance was found to be reversible by backflushing the xylem. Autoradiography showed that very large molecules do not pass through pit membrane pores. A comparison of pit membrane pore sizes to molecule sizes suggests that increased resistance to xylem flow is a result of plugging pit membrane pores. It was also found that pit membranes located in two parts of the plant differ in the apparent diameter of their pores and, thus, in their susceptibility to plugging by macromolecules. Macromolecules in xylem fluid may result from hostparasite interactions and may play a significant role in the outcome of the interaction.