Eugene J. Ford

Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia nigriscans

Munumbicins A, B, C and D are newly described antibiotics with a wide spectrum of activity against many human as well as plant pathogenic fungi and bacteria, and a Plasmodium sp. These compounds were obtained from Streptomyces NRRL 3052, which is endophytic in the medicinal plant snakevine (Kennedia nigriscans), native to the Northern Territory of Australia. This endophyte was cultured, the broth was extracted with an organic solvent and the contents of the residue were purified by bioassay-guided HPLC. The major components were four functionalized peptides with masses of 1269.6, 1298.5, 1312.5 and 1326.5 Da. Numerous other related compounds possessing bioactivity, with differing masses, were also present in the culture broth extract in lower quantities. With few exceptions, the peptide portion of each component contained only the common amino acids threonine, aspartic acid (or asparagine), glutamic acid (or glutamine), valine and proline, in varying ratios. The munumbicins possessed widely differing biological activities depending upon the target organism. For instance, munumbicin B had an MIC of 2.5 microg x ml(-1) against a methicillin-resistant strain of Staphylococcus aureus, whereas munumbicin A was not active against this organism. In general, the munumbicins demonstrated activity against Gram-positive bacteria such as Bacillus anthracis and multidrug-resistant Mycobacterium tuberculosis. However, the most impressive biological activity of any of the munumbicins was that of munumbicin D against the malarial parasite Plasmodium falciparum, having an IC(50) of 4.5+/-0.07 ng x ml(-1). This report also describes the potential of the munumbicins in medicine and agriculture.

Isopestacin, an isobenzofuranone from Pestalotiopsis microspora, possessing antifungal and antioxidant activities.

Isopestacin is an isobenzofuranone obtained from the endophytic fungus Pestalotiopsis microspora. While a few other isobenzofuranones are known from natural sources, isopestacin is the only one having a substituted benzene ring attached at the C-3 position of the furanone ring. The compound was isolated from culture broths of the fungus and crystallized and its structure was determined by X-ray crystallography. Both proton and carbon NMR spectral assignments are also reported for isopestacin. This compound possesses antifungal activity and, as measured by electron spin resonance specroscopy, it also behaves as an antioxidant scavenging both superoxide and hydroxy free radicals.

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.

Pestacin: a 1,3-dihydro isobenzofuran from Pestalotiopsis microspora possessing antioxidant and antimycotic activities

Abstract Pestalotiopsis microspora, an endophytic fungus native to the rainforest of Papua New Guinea, produces a 1,3-dihydro isobenzofuran. This product, pestacin, is 1,5,7-trisubstituted and exhibits moderate antifungal properties and antioxidant activity 11 times greater than the vitamin E derivative trolox. Antioxidant activity is proposed to arise primarily via cleavage of an unusually reactive C–H bond and, to a lesser extent, through O–H abstraction. Isolation of pestacin was achieved by extraction of culture fluid with methylene chloride followed by silica gel chromatography. Structure was established by X-ray diffraction and 13C and 1H NMR. The X-ray data demonstrate that pestacin occurs naturally as a racemic mixture. A mechanism for post-biosynthetic racemization is proposed.

Taxol from fungal endophytes and the issue of biodiversity

Fungi represent one of the most understudied and diverse group of organisms. Commonly, these organisms make associations with higher life forms and may proceed to biochemically mimic the host organism. An excellent example of this is the anticancer drug, taxol, which had been previously supposed to occur only in the plant genusTaxus (yew). However, taxol has been reported in a novel endophytic fungus—Taxomyces andreanae, but also has been demonstrated to occur in a number of unrelated fungal endophytes includingPestalotia, Pestalotiopsis, Fusarium, Alternaria, Pithomyces, Monochaetia and others. Thus, this report presents information on the presence of taxol among disparate fungal genera, and uses these observations as an additional argument to support efforts to study fungal endophytes and preserve their associated host plants.

Kakadumycins, novel antibiotics from Streptomyces sp. NRRL 30566, an endophyte of Grevillea pteridifolia

An endophytic streptomycete (NRRL 30566) is described and partially characterized from a fern-leaved grevillea (Grevillea pteridifolia) tree growing in the Northern Territory of Australia. This endophytic streptomycete produces, in culture, novel antibiotics - the kakadumycins. Methods are outlined for the production and chemical characterization of kakadumycin A and related compounds. This antibiotic is structurally related to a quinoxaline antibiotic, echinomycin. Each contains, by virtue of their amino acid compositions, alanine, serine and an unknown amino acid. Other biological, spectral and chromatographic differences between these two compounds occur and are given. Kakadumycin A has wide spectrum antibiotic activity, especially against Gram-positive bacteria, and it generally displays better bioactivity than echinomycin. For instance, against Bacillus anthracis strains, kakadumycin A has minimum inhibitory concentrations of 0.2-0.3 microg x ml(-1) in contrast to echinomycin at 1.0-1.2 microg x ml(-1). Both echinomycin and kakadumycin A have impressive activity against the malarial parasite Plasmodium falciparum with LD(50)s in the range of 7-10 ng x ml(-1). In macromolecular synthesis assays both kakadumycin A and echinomycin have similar effects on the inhibition of RNA synthesis. It appears that the endophytic Streptomyces sp. offer some promise for the discovery of novel antibiotics with pharmacological potential.

The induction of taxol production in the endophytic fungus-Periconia sp. from Torreya grandifolia

A Periconia sp was isolated from Torreya grandifolia (a relative of yew that does not synthesize taxol) near Huangshan National Park in the People’s Republic of China. This fungus, not previously known as a tree endophyte, was isolated from the inner bark of a small lower limb. When freshly isolated from the tree and placed in a semi-synthetic medium, the fungus produced readily detectable quantities of the anticancer drug taxol. Other taxol-producing endophytes were also isolated from this source. The production of taxol by Periconiasp was demonstrated unequivocally via spectroscopic and immunological methods. However, successive transfers of the fungus in semi-synthetic medium resulted in gradual attenuation until low production occurred even though fungal growth was relatively unaffected. Several compounds, known previously as activators of microbial metabolism, including serinol, p-hydroxybenzoic acid, and a mixture of phenolic acids, were capable of fully or partially restoring taxol production to otherwise taxol-attenuated cultures. The compound with the most impressive ability to activate taxol production was benzoic acid at 0.01 mM. Benzoic acid was not a taxol precursor.

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.

Heterokaryon formation and vegetative compatibility in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum was found to form stable heterokaryons. Auxotrophic mutants derived from six prototrophic wild-type strains were paired in all combinations on minimal media. Some combinations of auxotrophs led to the development of prototrophic strains. These prototrophic strains were demonstrated to be heterokaryons by separating the two parental nuclear types into separate thalli. Heterokaryon formation was under the control of a regulatory system that resulted in incompatibility in some combinations of strains, but not in others. Each strain was placed into one of three vegetative compatibility groups based on heterokaryon formation with the five other strains. Members belonging to one vegetative compatibility group came from different geographical locations, different hosts and were collected at disparate times. Vegetative compatibility groups did not directly correspond to mycelial compatibility groups.

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.