introduction to endophytes and their application to develop commercial products
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Ponencia: Introduction to endophytes and their application to develop commercial products Autor: Dr. Gary Strobel Evento TF Innova: Workshop Biotechnology "Isolation and identification of endophytic fungi from vascular plants"TRANSCRIPT
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The Promise of Endophyte Biology
Montana State University
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The Causal Agent of Dutch Elm Disease – Ceratocystis ulmi
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European Bark Beetle
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Antibiosis Effects of the Pseudomycins
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Transformation Plasmid
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The Field Experiment
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Systemic Candida albicans Infection in a Child
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Control of Black Sigatoka with Pseudomycin B
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Taxomyces andreanae
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Taxomyces andreanae an Endophyte on Yew
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Thin Layer Chromatography of Fungal Taxol and Authentic Taxol
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Electrospray MS of Fungal Taxol
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Products from the
RainforestIpecac
Taxol
Reserpine
Tubocurarine
Vinblastine
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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
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An Endophyte from the Jurassic Period
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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?
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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
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Selecting Plants for the Isolation of Endophytes
1.Visiting Areas of High Plant Biodiversity2. Finding Gondwanaland Plants3.Meeting and Learning from Native Peoples
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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
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A Bastion of Biodiversity-the World’s Rainforests
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Avenue of the giants
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The Spiny Forest of Southern Madagascar
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Angel Falls in the Tepui Lands of Venezuela
Highest in the world- 3212’
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Top of the Roraima Tepui
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A Novel Fungus
Seimatoantlerium
tepuiense
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Igauazu Falls-Brazil/Argentina
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An area of ancient Gondwanaland-Torres Del Paine, Chile
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Technique of Isolating Endophytes
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Some Endophytic fungi
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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.
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Cryptosporiopsis quercina
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Cryptosporiopsis spores
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HPLC Separation of Cryptocandin using an Altima C-18column
Bioactivity
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Cryptocandin MW 1079Pneumocandin MW 1079
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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
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Crystals of Cryptocin
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X-ray Crystallographic Device
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Structure of Cryptocin
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Carrao River
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Rhyncholacis penicillata under a water fall on the Carrao River
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Serratia marcescens on the plant surface
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Serratia marcescens Activity Against Pythium
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The Papuans
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Pestalotiopsis jesterii, a fungal endophyte
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Conidiospores of P. jesterii
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Ambuic Acid is produced by various isolates of Pestalotiopsis microsporaobtained in various rainforest locations around the world
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Monstera speciosa in the Jungles of the Upper Amazon of Peru
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P-25 An Endophytic Streptomycete from the Monstera vine
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Origin of Spores in P-25 vs. Penicillum sp.
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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
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Activity of P-25 Against Pythium
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Anopheles sp. vector of Plasmodium sp.- cause of Malaria
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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
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My Native Australian Friends
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Northern Territory Australia – a Monsoonal Rainforest
Snakevine-Kennedianigriscans
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Streptomyces sp. A-11 from Snakevine
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HPLC Separation of the Components of Streptomyces sp. A-11
Actinomycin X2
Actinomycin D
Actinomycin X0 beta
Munumbicins E-4,5M.W. 1315 & 1431
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Some Representative Streptomycetes fromSnakevine
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New, Very Unusual Streptomycetes spp. from Snakevine
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Streptomycete 149 -- Horn Structure
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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.
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