Gary A. Strobel

Bioprospecting for Microbial Endophytes and Their Natural Products

SUMMARY Endophytic microorganisms are to be found in virtually every plant on earth. These organisms reside in the living tissues of the host plant and do so in a variety of relationships, ranging from symbiotic to slightly pathogenic. Because of what appears to be their contribution to the host plant, the endophytes may produce a plethora of substances of potential use to modern medicine, agriculture, and industry. Novel antibiotics, antimycotics, immunosuppressants, and anticancer compounds are only a few examples of what has been found after the isolation, culture, purification, and characterization of some choice endophytes in the recent past. The potential prospects of finding new drugs that may be effective candidates for treating newly developing diseases in humans, plants, and animals are great.

Endophytes as sources of bioactive products

An increase in the number of people in the world having health problems caused by various cancers, drug-resistant bacteria, parasitic protozoans, and fungi is a cause for alarm. An intensive search for newer and more effective agents to deal with these disease problems is now under way and endophytes are a novel source of potentially useful medicinal compounds.

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.

Volatile antimicrobials from Muscodor crispans ,a novel endophytic fungus

Muscodor crispans is a recently described novel endophytic fungus of Ananas ananassoides (wild pineapple) growing in the Bolivian Amazon Basin. The fungus produces a mixture of volatile organic compounds (VOCs); some of the major components of this mixture, as determined by GC/MS, are propanoic acid, 2-methyl-, methyl ester; propanoic acid, 2-methyl-; 1-butanol, 3-methyl-;1-butanol, 3-methyl-, acetate; propanoic acid, 2-methyl-, 2-methylbutyl ester; and ethanol. The fungus does not, however, produce naphthalene or azulene derivatives as has been observed with many other members of the genus Muscodor. The mixture of VOCs produced by M. crispans cultures possesses antibiotic properties, as does an artificial mixture of a majority of the components. The VOCs of the fungus are effective against a wide range of plant pathogens, including the fungi Pythium ultimum, Phytophthora cinnamomi, Sclerotinia sclerotiorum and Mycosphaerella fijiensis (the black sigatoka pathogen of bananas), and the serious bacterial pathogen of citrus, Xanthomonas axonopodis pv. citri. In addition, the VOCs of M. crispans killed several human pathogens, including Yersinia pestis, Mycobacterium tuberculosis and Staphylococcus aureus. Artificial mixtures of the fungal VOCs were both inhibitory and lethal to a number of human and plant pathogens, including three drug-resistant strains of Mycobacterium tuberculosis. The gaseous products of Muscodor crispans potentially could prove to be beneficial in the fields of medicine, agriculture, and industry.

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.

Involvement of a plasmid in the hairy root disease of plants caused by Agrobacterium rhizogenes

Abstract Agrobacterium rhizogenes causes a proliferation of roots on plants that it infects. This is in contrast to Agrobacterium tumefaciens which causes gall or tumor formation on its hosts. A large molecular weight plasmid (1.1 × 108) in A. rhizogenes strain A4 is correlated with the infectivity of this organism. However, this plasmid apparently carries additional information not vital to the infection process. Experimental evidence supporting these conclusions is: (i) A. rhizogenes A4 loses infectivity when all or part of the plasmid is lost after treatment with ethidium bromide or after heating at 37 °C. (ii) There occurs successful conjugational transfer of the A4 plasmid in planta to a noninfectious, antibiotic-resistant A. radiobacter. Infectious transconjugants were antibiotic resistant and contain a plasmid comparable to that of A. rhizogenes A4. (iii) A. rhizogenes A4 and the transconjugants possessed identical EcoR1 restriction endonuclease patterns, whereas three ethidium bromide-treated isolates that were noninfectious but plasmid containing had lost or gained bands in the pattern. The infectious plasmid of A. rhizogenes A4 has been designated pHrA4. Some potential benefits of the A. rhizogenes plasmid to agriculture are discussed.

Rainforest Endophytes and Bioactive Products

ABSTRACT:  An increase in the number of people in the world having health problems caused by certain cancers, drug-resistant bacteria, parasitic protozoans, and fungi has caused alarm. An intensive search for newer and more effective agents to deal with these problems is now underway. Endophytes are a potential source of novel chemistry and biology to assist in helping solve not only human health, but plant and animal health problems also. Endophytes reside in the tissues between living plant cells. The relationship that they establish with the plant varies from symbiotic to bordering on pathogenic. Of all of the worlds plants, it seems that only a few grass species have had their complete complement of endophytes studied. As a result, the opportunity to find new and interesting endophytes among the myriad of plants is great. Sometimes extremely unusual and valuable organic substances are produced by these endophytes. These compounds may contribute to the host-microbe relationship. The initial step in dealing with endophytic microorganisms is their successful isolation from plant materials. Then, the isolation and characterization of bioactive substances from culture filtrates is done using bioassay guided fractionation and spectroscopic methods. Some of the more interesting compounds produced by endophytic microbes with which we have dealt are taxol, cryptocin, cryptocandin, jesterone, oocydin, isopestacin, the pseudomycins and ambuic acid. This review discusses an approach for bio-prospecting the rainforests, not only to harvest their endophytic microorganisms, but to eventually build a better understanding of the importance and value they have to humankind.

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.