The Promise of Endophyte Biology

Montana State University

The Causal Agent of Dutch Elm Disease – Ceratocystis ulmi

European Bark Beetle

Antibiosis Effects of the Pseudomycins

Transformation Plasmid

The Field Experiment

Systemic Candida albicans Infection in a Child

Control of Black Sigatoka with Pseudomycin B

Taxomyces andreanae

Taxomyces andreanae an Endophyte on Yew

Thin Layer Chromatography of Fungal Taxol and Authentic Taxol

Electrospray MS of Fungal Taxol

Products from the

RainforestIpecac

Taxol

Reserpine

Tubocurarine

Vinblastine

Definition of An Endophyte

By definition an endophyte is amicroorganism that becomes established in a higherplant and does not cause any overt symptoms or cause any apparent tissue damage

An Endophyte from the Jurassic Period

Areas of High Plant Biodiversity

70-80% of the Earth’s Plant Diversity Occurs on 1.5% of Its Land Area

Is this also true of microbial diversity?

Plant Diversity in the Canary Isles30 million year old landscape..

1.About 2000 plant species2.About 528 of these are endemic3.Chances of finding novel endophytes

are great

Selecting Plants for the Isolation of Endophytes

1.Visiting Areas of High Plant Biodiversity2. Finding Gondwanaland Plants3.Meeting and Learning from Native Peoples

Progress on Endophytic MicrobesExamples of products-jesterone, hydroxyjesteronepestacin, isopestacin, ambuic acid, ecomycin, oocydin A munumbicins, kakadumycin, cryptocandin, pseudomycins and others.

Ambuic Acid: JOC (2003) 4609-4614

A Bastion of Biodiversity-the World’s Rainforests

Avenue of the giants

The Spiny Forest of Southern Madagascar

Angel Falls in the Tepui Lands of Venezuela

Highest in the world- 3212’

Top of the Roraima Tepui

A Novel Fungus

Seimatoantlerium

tepuiense

Igauazu Falls-Brazil/Argentina

An area of ancient Gondwanaland-Torres Del Paine, Chile

Technique of Isolating Endophytes

Some Endophytic fungi

Tripterigeum wilfordii a Chinese medicinal plant used to treat arthritis and other ailments of the joints and limbs possesses a plethora of endophytic microbes including Cryptosporiopsis quercina.

Cryptosporiopsis quercina

Cryptosporiopsis spores

HPLC Separation of Cryptocandin using an Altima C-18column

Bioactivity

Cryptocandin MW 1079Pneumocandin MW 1079

Test Fungal

Pathogen

MIC

micrograms/ml

of Cryptocandin

MIC of

Amphotericin B

Candida albicans 0.03 0.03

Cryptococcus

neoformans > 20.0 0.04

Aspergillus

fumigatus >20.0 0.03

Histoplasma

capsulatum 0.01 0.01

Biological Activity of Cryptocandin

Crystals of Cryptocin

X-ray Crystallographic Device

Structure of Cryptocin

Carrao River

Rhyncholacis penicillata under a water fall on the Carrao River

Serratia marcescens on the plant surface

Serratia marcescens Activity Against Pythium

The Papuans

Pestalotiopsis jesterii, a fungal endophyte

Conidiospores of P. jesterii

Ambuic Acid is produced by various isolates of Pestalotiopsis microsporaobtained in various rainforest locations around the world

Monstera speciosa in the Jungles of the Upper Amazon of Peru

P-25 An Endophytic Streptomycete from the Monstera vine

Origin of Spores in P-25 vs. Penicillum sp.

Some Characteristics of Isolate P-251. Is endophytic on Monstera speciosa2. Appears to be a streptomycete

a. Only bacterial primers functionto give product from its ribosomal DNA

b. Primers for 18Sr DNA do not functionc. Has no discernable nucleus (by nuclear staining)d. Gram positive

3. Has 16S r DNA sequences that have 95% similarity to Streptoverticillum sp.

4. Makes Coronamycins4. Has some characteristics of

Penicillum sp. but origin of spores is unique5. Is not inhibited by cyclohexamide up to (or

greater) than 100 ppm

Activity of P-25 Against Pythium

Anopheles sp. vector of Plasmodium sp.- cause of Malaria

Properties of Coronamycins

1.Peptides- 2 principle ones MW.’s 1217 and 1203

2. Composition x:x methionine:tyrosine and leucine-ratio-- 2:2:1:1:3

3. Active against pythicaeous fungi (2-16 micrograms/ml) ,and Cryptococcus neoformans.

4. Extremely active against Plasmodium falciparum IC 50 <1 ng /ml

My Native Australian Friends

Northern Territory Australia – a Monsoonal Rainforest

Snakevine-Kennedianigriscans

Streptomyces sp. A-11 from Snakevine

HPLC Separation of the Components of Streptomyces sp. A-11

Actinomycin X2

Actinomycin D

Actinomycin X0 beta

Munumbicins E-4,5M.W. 1315 & 1431

Some Representative Streptomycetes fromSnakevine

New, Very Unusual Streptomycetes spp. from Snakevine

Streptomycete 149 -- Horn Structure

Rainforest Microbiology- The Prospects and Promises

1. Each individual plant has one or more associated microorganisms. Only a few microbes have ever been studied.

2. The relationships of the plant to the microbe may range from symbiotic to weakly pathogenic.

3. Host specificity may exist with certain microbes which places a huge emphasis on the preservation of the world’s rainforests.

4. Products from endophytes have a wide range of uses in medicine, agriculture and industry.

5. Peculiar microbes are plentiful and usually are to be found in areas of high biodiversity.

6. The biology of endophytes is largely unknown.