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"

1. Montana State University
The Promise of Endophyte Biology

3. The Causal Agent of Dutch Elm Disease – Ceratocystis ulmi

4. European Bark Beetle

5. Antibiosis Effects of the Pseudomycins

6. Transformation Plasmid

7. The Field Experiment

11. Systemic Candida albicans Infection in a Child

12. Control of Black Sigatoka with Pseudomycin B

13. Taxomyces andreanae

14. Taxomyces andreanae an Endophyte on Yew

16. Thin Layer Chromatography of Fungal Taxol and
Authentic Taxol

17. Electrospray MS of Fungal Taxol

19. Ipecac Products from the
Rainforest
Taxol
Vinblastine
Reserpine
Tubocurarine

20. Definition of An Endophyte
By definition an endophyte is a
microorganism that becomes
established in a higher
plant and does not cause any
overt symptoms or cause any
apparent tissue damage

21. An Endophyte from the Jurassic Period

22. 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?

23. ant Diversity in the Canary Isles
0 million year old landscape..
About 2000 plant species
About 528 of these are endemic
Chances of finding novel endophytes
are great

24. Selecting Plants for the Isolation of
Endophytes
1. Visiting Areas of High Plant Biodiversity
2. Finding Gondwanaland Plants
3.Meeting and Learning from Native Peoples

25. Progress on Endophytic Microbes
Examples of products-jesterone, hydroxyjesterone
pestacin, isopestacin, ambuic acid, ecomycin, oocydin A
munumbicins, kakadumycin, cryptocandin, pseudomycins
and others.
Ambuic Acid: JOC (2003) 4609-4614

26. A Bastion of Biodiversity-the
World’s Rainforests

27. Avenue of the giants

28. The Spiny Forest of Southern Madagascar

29. Angel Falls in the Tepui Lands of Venezuela
Highest in the world- 3212’

30. Top of the Roraima Tepui

31. A Novel Fungus
Seimatoantlerium
tepuiense

32. Igauazu Falls-Brazil/Argentina

33. An area of ancient Gondwanaland-Torres Del Paine, Chile

34. Technique of Isolating Endophytes

35. Some
Endophytic fungi

36. 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.

37. Cryptosporiopsis quercina

38. Cryptosporiopsis spores

39. HPLC Separation of Cryptocandin
using an Altima C-18column
Bioactivity

40. Cryptocandin MW 1079
Pneumocandin MW 1079

41. Biological Activity of Cryptocandin
Test Fungal MIC MIC of
Pathogen micrograms/ml Amphotericin B
of Cryptocandin
Candida albicans 0.03 0.03
Cryptococcus > 20.0 0.04
neoformans
Aspergillus >20.0 0.03
fumigatus
Histoplasma 0.01 0.01
capsulatum

42. Crystals of Cryptocin

43. X-ray Crystallographic Device

44. Structure of
Cryptocin

45. Carrao River

46. Rhyncholacis penicillata under a
water fall on the Carrao River

47. Serratia marcescens on the plant surface

48. Serratia marcescens Activity Against Pythium

50. The Papuans

51. Pestalotiopsis jesterii, a fungal endophyte

52. Conidiospores of P. jesterii

54. Ambuic Acid is
produced by
various isolates of
Pestalotiopsis
microspora
obtained in various
rainforest
locations around
the world

55. Monstera speciosa in the Jungles
of the Upper Amazon of Peru

56. P-25 An Endophytic
Streptomycete from the
Monstera vine

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

58. Some Characteristics of Isolate P-25
1. Is endophytic on Monstera speciosa
2. Appears to be a streptomycete
a. Only bacterial primers function
to give product from its ribosomal DNA
b. Primers for 18Sr DNA do not function
c. 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 Coronamycins
4. Has some characteristics of
Penicillum sp. but origin of spores is unique
5. Is not inhibited by cyclohexamide up to (or
greater) than 100 ppm

59. Activity of P-25 Against Pythium

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

61. 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

62. My Native Australian Friends

63. Northern Territory Australia – a Monsoonal
Rainforest
Snakevine-Kennedia
nigriscans

64. Streptomyces sp. A-11 from Snakevine

65. HPLC Separation of the Components of
Streptomyces sp. A-11
Munumbicins E-4,5
M.W. 1315 & 1431
Actinomycin D
Actinomycin X2
Actinomycin X0 beta

66. Some Representative Streptomycetes from
Snakevine

67. New, Very Unusual Streptomycetes spp. from
Snakevine

68. Streptomycete 149 -- Horn Structure

69. 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.