Ponencia: Instructions for the collection of plant specimens and endophytes studies Autor: Dr. Gary Strobel Evento TF Innova: Workshop Biotechnology "Isolation and identification of endophytic fungi from vascular plants"

1. The story of Muscodor albus

2. Avenue of the giants

3. Honduran Rainforest
With High Plant Diversity Comes High Microbial Diversity

4. Cinnamomum zeylanicum

5. Phytophageous Mites

6. Anti-Mite Measures

7. SEM of the fungus

8. Maximum Parsimony Phenogram of
18S r DNA Sequences of Xylariales

9. Muscodor albus
Properties
No spores
2.Ropy mycelium
3.Strange odor
4.Antibiotic activity US Patent-6911338
5.Related to xylaria

10. Bioassay of VOC’s of M. albus

11. Trapping the VOC’s of M. albus
Divinylbenzene/carburen on
polydimethylsiloxane on a stable flex fibre

12. Joe Sears with GC/MS

18. The VOC’s of M. albus

19. Bioassay of Fungal
VOC’s

20. Bioactivity of M. albus and its VOC‘s

21. Bioactivity of M. albus VOC’s
Test Organism Alcohols Esters Ketones Acids Lipids
0.48 l/CC 0.53 l/CC 0.02 l/CC 0.09 l/CC 0.08l/CC
% growth of % growth of % growth of % growth of % growth of
control control control control control
Pythium ultimum 11.2  4 0 67.5  7 40.9  3 75  0
Rhizoctonia solani 55 5 0 67.57.5 67.57.5 400
Tapesia yallundae 3515 0 75  25 100 0 1000
Xylaria sp. 7525 0 1000 1000 1000
Sclerotinia sclerotiorum 293 8.11.5 20.612 400 782
Cercospora beticola 588 55 1000 8317 1000
Fusarium solani 7010 55 5 9010 8020 8010

23. Proton Transfer Reaction Mass Spectrometer

24. The PTR – MS of Standard VOCs of M. albus
Compound Major ion and % in ( )
Acetic acid, 2-phenylethyl 43 (37), 61 (63)
ester
Phenyl ethyl alcohol 105 (100)
Propanoic acid, 2-methyl, 41 (7), 43 (18), 57 (35), 89 (31), 145 (9)
2-methylpropyl ester
Propanoic acid, 2-methyl, 43 (39), 71 (42), 89 (16), 159 (3)
3-methylbutyl ester
Propanoic acid, 2 methyl, 43 (4), 89 (11), 117 (85)
ethyl ester
Propanoic acid,2-methyl 41 (11), 43 (42), 89 (47)
Propanoic acid, 2-methyl, 43 (3), 103(97)
methyl ester
1-Butanol, 3-methyl, 41 (10), 43 (47), 71 (37), 131 (4)
acetate

25. 34
Temperature
800
33
mass 131
32
Temperature (C)
600
Intensity (cps)
31
400
30
mass 103 29
200
Mass 131= Ca. 18ppb 28
Mass 103=Ca. 12ppb
0
14 15 16 17 18 19
Time (days)

26. Treating Barley Seeds with M. albus

27. Control of Loose Smut by M. albus
Ustilago hordei on barley

28. Protective effects of M. albus

30. Preserving Garbage with M. albus VOCs

31. Tons of Muscodor albus

32. Muscodor albus in action- decontaminating
human waste products

33. Phillips Toilet on the North Col of Mt Everest

34. Access to the Upper Amazon of Peru

35. M. vitigenes
from Paullinia
paullinioides

36. Muscodor roseus from Ironwood in Australia

37. S.E Asia
Tesso Nilo collecting site

38. Collecting in the Tesso Nilo Area of
Sumatra, Indonesia

39. M. albus Isolate I-41 Sumatra, Indonesia

40. Coastal Ecuador- Dry Forest

41. Fun in the jungle

42. A Jungle Nasty-fer de Lance
Percy’s foot

43. M. albus from Guazuma
ulmifolia in the dry forest
Of coastal Ecuador

44. Table 1. GC/MS analysis of the volatile compounds
produced by M. albus E-6.
RT Total M/z Possible compound MW
Area
(%)
4:58 32.4 102 *Propanoic acid, 2- 102
methyl-methyl ester
7:07 1.2 116 Butanoic acid, 2-methyl- 116
7:24 1.0 116 Butanoic acid, 3-methyl- 116
9:35 1.3 84 2-Butenal, 2-methyl- 84
10:19 28.0 130 *1 Butanol, 2-methyl- 130
12:20 5.9 158 Butanoic acid, 3- 158
methylbutyl ester
12:24 4:2 158 *Propanoic acid, 2- 158
methyl, 2 -methylbutyl
ester
13:51 1.1 118 Propanoic acid, 2- 118
hydroxy-2-methyl-methyl
ester
14:07 1.0 86 3-Buten-1-ol, 3-methyl- 86
15:36 1.6 140 1-Octene, 3-ethyl- 140
16:12 1.1 142 *4-Nonanone 142
18:21 1.4 204 Naphthalene,decahydro- 204
4a-methyl-1-methylene-7-
(1methylethylidine)-,(4aR-
trans)
19:54 1.0 204 1H- 204
cycloprop[e]azulene,1a,2,
3,5,6,7,7a,7b-octahydro-
1,1,4,7-tetramethyl-,[1aR-
(1a.alpha.)]
20:02 3.8 222 Guaiol 222
20:25 5.7 204 *Caryophyllene 204
22:33 100 88 *Propanoic acid, 2- 88
methyl-
24:36 1.8 101 Formamide,N-(1- 101
methylpropyl)
24:55 1.0 98 2-Furanmethanol 98
25:57 1.0 204 *Azulene, 1,2,3,5,6,7,8, 204

45. Muscodor crispans. from
Ananas ananasoides
(Bolivan Amazon)

46. Retention Time Min. Total Area Possible Compound M- Da
2:05 0.139 Acetaldehyde 44.03
VOCs 3:40
3:51
0.623
0.283
Ethyl Acetate
2-Butanone
88.05
72.06
B-23 4:08 3.056 Propanoic acid, 2-methyl-,
methyl ester
102.07
4:18 1.241 Ethanol 46.04
5:29 0.229 Acetic acid, 2-methylpropyl 116.08
ester
6:39 0.109 Propanoic acid, 2-methyl-, 2- 144.12
methylpropyl ester
6:46 0.178 1-Propanol, 2-methyl- 74.07
6:52 0.151 2-Butenal, 2-methyl-, (E)- 84.06
7:12 0.479 1-Butanol, 3-methyl-, acetate 130.10
8:18 0.301 Hexane, 2,3-dimethyl- 114.14
8:21 0.478 Propanoic acid, 2-methyl-, 2- 158.13
methylbutyl ester
8:31 1.538 1-Butanol, 3-methyl- 88.09
13:37 35.118 Propanoic acid, 2-methyl- 88.05
14:41 0.394 Formamide, N-(1- 101.08
methylpropyl)-
16:44 0.131 Acetic acid, 2-phenylethyl 164.08
ester
20:44 0.720 Cyclohexane, 1,2-dimethyl- 192.19
3,5-bis(1-methylethenyl)-

47. Effects of B-23 on human pathogenic bacteria
Organism Type of Exposure Growth/ No Comments
Cell Wall Time Growth
(in the presence of M.
crispans)
S. aureus 6538 Gram + 2, 4 and 6 No growth
days
S. cholerasuis 10708 Gram - 2, 4 and 6 No growth
days
P. aeruginosa 15442 Gram - 2 days Growth No visible difference between
exposed and control plates.
S. aureus ATCC 43300 Gram + 2, 4 and 6 Growth No actual colonies formed,
(MRSA) days just a slightly filmy growth.
Y. pestis 91-3365 Gram - 3 and 5 days No growth
B. anthracis A2084 Gram + 3 and 5 days Growth Only a few colonies left after
exposure and when
incubated, more grew.
M. tuberculosis 3081 Acid-fast 2, 4, 7 and 14 No growth
(resistant to isoniazid) days
M. tuberculosis 50001106 Acid-fast 2, 4, 7 and 14 No growth
(resistant to streptomycin) days
M. tuberculosis 59501228 Acid-fast 2, 4, 7 and 14 No growth
(resistant to days
streptomycin/ethambutol)
M. tuberculosis 59501867 Acid-fast 2, 4, 7 and 14 No growth
(susceptible) days

48. Distribution of Muscodor spp. in the World

49. Plant (Family)Sources of Muscodor albus
1. Lauraceae
2. Myristicaceae
3. Proteaceae
4. Combretaceae
5. Sapindaceae
6. Leguminosae
7. Caesalpiniaceae
8. Bromeliaceae

50. Lessons Learned from M. albus and its
relatives
1. Each has 95% -99% 18S rDNA partial sequence
similarity to the original isolate of M. albus -620.
2. They make different VOC’s and in differing amounts.
3. They are found as endophytes in the wet tropical
regions of the world from Thailand, to Indonesia,
Australia, Venezuela, and Peru. They are confined to +/-
16 degrees from the equator.
4, Each has some type of bioactivity.
5. Some are being developed for commercial purposes.
6. Many other surprises remain in the jungle.

51. Northern Patagonia –Land of the Alerces

52. Torres del Paine

53. Calving of a glacier in a hanging glacier-Chile

54. Ulmo and Northern Patagonia

55. An Endophytic Gliocladium sp. from Eucryphia
cordifolia in Northern Patagonia
Isolated by the M. albus selection
technique

56. SEM’s of Gliocladium sp.

57. The Hydrocarbons of Gliocladium sp.
Relati Molecu Grown on Oat meal agar 18 days under minimal oxygen
ve Possible lar
Time Area Compound Weight
1.603 1.213 Oxirane, ethyl- 72.06 7.648 1.807 1-Butanol, 3-methyl-, acetate 130.10
2.081 1.419 Heptane, 2-methyl- 114.14 7.836 1.928 2-n-Butyl furan 124.09
2.666 2.519 Octane 114.14 8.026 0.279 Benzene, 1 3-dimethyl- 106.08
3.138 0.261 1-Octene 112.13 8.114 0.368 Decane, 3, 3, 5-trimethyl- 184.22
4.598 7.132 Ethanol 46.04 8.303 0.335 Pentane, 1-iodo- 197.99
4.872 1.133 Cyclohexene, 4-methyl- 96.09 8.364 1.379 2-Hexanol 102.10
5.204 0.342 Hexane, 2, 4-dimethyl- 114.14 8.498 0.306 Acetic acid, pentyl ester 130.10
5.378 0.180 Undecane, 2, 6-dimethyl- 184.22 8.735 1.228 Hexanoic acid, methyl ester 130.10
5.533 0.504 Hexadecane 226.27 9.066 7.956 1-Butanol, 3-methyl- 88.09
Heptane, 5-ethyl-2, 2, 3- 9.302 0.134 Phenol, 4-ethyl- 122.07
5.941 0.564 trimethyl- 170.20
9.817 0.710 3-Octanone 128.12
6.365 0.476 Undecane, 4-methyl- 170.20
10.054 1.780 Acetic acid, hexyl ester 144.12
Heptane, 5-ethyl-2, 2, 3-
6.418 0.180 trimethyl- 170.20 10.708 0.143 2-Heptanol 116.12
Octane, 3-ethyl-2, 7- 10.985 0.574 7-Octen-2-one 126.10
6.668 0.155 dimethyl- 170.20 11.242 6.514 Cyclopropane, propyl- 84.09
6.768 1.021 Decane, 2, 2, 6-trimethyl- 184.22 11.329 0.550 Acetic acid, sec-octyl ester 172.15
6.931 0.360 Undecane 156.19 11.545 11.294 Acetic acid, heptyl ester 158.13
7.112 0.195 Decane, 3, 3, 5-trimethyl- 184.22 11.775 0.205 Octanoic acid, methyl ester 158.13
7.173 0.592 Nonane, 3-methyl- 142.17 11.938 0.485 3, 5-Octadiene (Z, Z) 110.11
7.232 0.601 1-Propanol, 2-methyl- 74.07 12.265 3.289 1-Butanol, 3-methyl-, acetate 131.11
7.325 0.746 Furan, 4-methyl-2-propyl- 124.09

58. Hydrocarbons ..continued
12.878 11.533 Acetic acid 60.02
12.931 12.008 Acetic acid, octyl ester 172.15
13.381 0.222 3-Octen-2-ol (Z) 128.12
13.584 0.176 Neoisolongifolene 204.19
14.455 0.130 Propanic acid, 2-methyl- 88.05
14.543 0.134 Cycloheptanone, 2-methylene- 124.09
14.651 0.651 1H-Indene, octahydro-, cis 124.13
Cyclopentadiene, 5, 5-dimethyl-1-
14.926 0.254 ethyl- 122.11
15.335 0.137 Butyrolactone 86.04
15.477 0.147 Acetic acid, decyl ester 200.18
15.673 0.465 Pentanoic acid, 3-methyl- 116.08
15.835 0.271 Cyclodecene 138.14
16.472 0.174 Pentanoic acid 102.07
17.653 1.657 Hexanoic acid 116.08
18.360 1.073 Phenylethyl alcohol 122.07
19.588 0.355 Phenol, 4-ethyl-2methoxy- 152.08

60. Gliocladium a virtual chemistry factory

61. Victoria, Australia
temperate rainforest

62. The stages of Ascocoryne

63. The Victoria Fires of 09

64. Could fungi have contributed to the formation
of crude oil ??
SEM’s of Gliocladium sp. and a fossilized fungus

65. Chief Contributors