instructions for the collection of plant specimens and endophytes studies
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Ponencia: Instructions for the collection of plant specimens and endophytes studiesAutor: Dr. Gary StrobelEvento TF Innova: Workshop Biotechnology "Isolation and identification of endophytic fungi from vascular plants"TRANSCRIPT
The story of Muscodor albus
Avenue of the giants
Honduran Rainforest
With High Plant Diversity Comes High Microbial Diversity
Cinnamomum zeylanicum
Phytophageous Mites
Anti-Mite Measures
SEM of the fungus
Maximum Parsimony Phenogram of 18S r DNA Sequences of Xylariales
Muscodor albus
US Patent-6911338
PropertiesNo spores2.Ropy mycelium3.Strange odor4.Antibiotic activity5.Related to xylaria
Bioassay of VOC’s of M. albus
Trapping the VOC’s of M. albus
Divinylbenzene/carburen on polydimethylsiloxane on a stable flex fibre
Joe Sears with GC/MS
The VOC’s of M. albus
Bioassay of Fungal VOC’s
Bioactivity of M. albus and its VOC‘s
Bioactivity of M. albus VOC’s
Test Organism Alcohols
0.48 l/CC
% growth of
control
Esters
0.53 l/CC
% growth of
control
Ketones
0.02 l/CC
% growth of
control
Acids
0.09 l/CC
% growth of
control
Lipids
0.08l/CC
% growth of
control
Pythium ultimum 11.2 4 0 67.5 7 40.9 3 75 0
Rhizoctonia solani 55 5 0 67.57.5 67.57.5 400
Tapesia yallundae 3515 0 75 25 100 0 1000
Xylaria sp. 7525 0 1000 1000 1000
Sclerotinia sclerotiorum 293 8.11.5 20.612 400 782
Cercospora beticola 588 5 5 1000 8317 1000
Fusarium solani 7010 55 5 9010 8020 8010
Proton Transfer Reaction Mass Spectrometer
Compound Major ion and % in ( )
Acetic acid, 2-phenylethyl
ester
43 (37), 61 (63)
Phenyl ethyl alcohol 105 (100)
Propanoic acid, 2-methyl,
2-methylpropyl ester
41 (7), 43 (18), 57 (35), 89 (31), 145 (9)
Propanoic acid, 2-methyl,
3-methylbutyl ester
43 (39), 71 (42), 89 (16), 159 (3)
Propanoic acid, 2 methyl,
ethyl ester
43 (4), 89 (11), 117 (85)
Propanoic acid,2-methyl 41 (11), 43 (42), 89 (47)
Propanoic acid, 2-methyl,
methyl ester
43 (3), 103(97)
1-Butanol, 3-methyl,
acetate
41 (10), 43 (47), 71 (37), 131 (4)
The PTR – MS of Standard VOCs of M. albus
800
600
400
200
0
Inte
nsity (
cp
s)
191817161514
Time (days)
34
33
32
31
30
29
28
Te
mp
era
ture
(C
)
Temperature
mass 103
mass 131
Mass 131= Ca. 18ppb
Mass 103=Ca. 12ppb
Treating Barley Seeds with M. albus
Control of Loose Smut by M. albus
Ustilago hordei on barley
Protective effects of M. albus
Preserving Garbage with M. albus VOCs
Tons of Muscodor albus
Muscodor albus in action- decontaminating
human waste products
Phillips Toilet on the North Col of Mt Everest
Access to the Upper Amazon of Peru
M. vitigenes
from Paullinia
paullinioides
Muscodor roseus from Ironwood in Australia
S.E Asia
Tesso Nilo collecting site
Collecting in the Tesso Nilo Area ofSumatra, Indonesia
M. albus Isolate I-41 Sumatra, Indonesia
Coastal Ecuador- Dry Forest
Fun in the jungle
Percy’s foot
A Jungle Nasty-fer de Lance
M. albus from Guazumaulmifolia in the dry forest Of coastal Ecuador
Table 1. GC/MS analysis of the volatile compounds produced by M. albus E-6. RT Total
Area (%)
M/z Possible compound MW
4:58 32.4 102 *Propanoic acid, 2-methyl-methyl ester
102
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-
methylbutyl ester 158
12:24 4:2 158 *Propanoic acid, 2-methyl, 2 -methylbutyl
ester
158
13:51 1.1 118 Propanoic acid, 2-hydroxy-2-methyl-methyl
ester
118
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-
4a-methyl-1-methylene-7-(1methylethylidine)-,(4aR-
trans)
204
19:54 1.0 204 1H-cycloprop[e]azulene,1a,2,3,5,6,7,7a,7b-octahydro-1,1,4,7-tetramethyl-,[1aR-
(1a.alpha.)]
204
20:02 3.8 222 Guaiol 222 20:25 5.7 204 *Caryophyllene 204 22:33 100 88 *Propanoic acid, 2-
methyl- 88
24:36 1.8 101 Formamide,N-(1-methylpropyl)
101
24:55 1.0 98 2-Furanmethanol 98 25:57 1.0 204 *Azulene, 1,2,3,5,6,7,8, 204
Muscodor crispans. from Ananas ananasoides (Bolivan Amazon)
Retention Time Min. Total Area Possible Compound M- Da
2:05 0.139 Acetaldehyde 44.03
3:40 0.623 Ethyl Acetate 88.05
3:51 0.283 2-Butanone 72.06
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
ester
116.08
6:39 0.109 Propanoic acid, 2-methyl-, 2-
methylpropyl ester
144.12
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-
methylbutyl ester
158.13
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-
methylpropyl)-
101.08
16:44 0.131 Acetic acid, 2-phenylethyl
ester
164.08
20:44 0.720 Cyclohexane, 1,2-dimethyl-
3,5-bis(1-methylethenyl)-
192.19
VOCs B-23
Organism Type of
Cell Wall
Exposure
Time
Growth/ No
Growth (in the presence of M.
crispans)
Comments
S. aureus 6538 Gram + 2, 4 and 6
days
No growth
S. cholerasuis 10708 Gram - 2, 4 and 6
days
No growth
P. aeruginosa 15442 Gram - 2 days Growth No visible difference between
exposed and control plates.
S. aureus ATCC 43300
(MRSA)
Gram + 2, 4 and 6
days
Growth No actual colonies formed,
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
(resistant to isoniazid)
Acid-fast 2, 4, 7 and 14
days
No growth
M. tuberculosis 50001106
(resistant to streptomycin)
Acid-fast 2, 4, 7 and 14
days
No growth
M. tuberculosis 59501228
(resistant to
streptomycin/ethambutol)
Acid-fast 2, 4, 7 and 14
days
No growth
M. tuberculosis 59501867
(susceptible)
Acid-fast 2, 4, 7 and 14
days
No growth
Effects of B-23 on human pathogenic bacteria
Distribution of Muscodor spp. in the World
Plant (Family)Sources of Muscodor albus
1. Lauraceae
2. Myristicaceae
3. Proteaceae
4. Combretaceae
5. Sapindaceae
6. Leguminosae
7. Caesalpiniaceae
8. Bromeliaceae
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.
Northern Patagonia –Land of the Alerces
Torres del Paine
Calving of a glacier in a hanging glacier-Chile
Ulmo and Northern Patagonia
An Endophytic Gliocladium sp. from Eucryphia cordifolia in Northern Patagonia
Isolated by the M. albus selection technique
SEM’s of Gliocladium sp.
Time
Relati
ve
Area
Possible
Compound
Molecu
lar
Weight
1.603 1.213 Oxirane, ethyl- 72.06
2.081 1.419 Heptane, 2-methyl- 114.14
2.666 2.519 Octane 114.14
3.138 0.261 1-Octene 112.13
4.598 7.132 Ethanol 46.04
4.872 1.133 Cyclohexene, 4-methyl- 96.09
5.204 0.342 Hexane, 2, 4-dimethyl- 114.14
5.378 0.180 Undecane, 2, 6-dimethyl- 184.22
5.533 0.504 Hexadecane 226.27
5.941 0.564
Heptane, 5-ethyl-2, 2, 3-trimethyl- 170.20
6.365 0.476 Undecane, 4-methyl- 170.20
6.418 0.180
Heptane, 5-ethyl-2, 2, 3-trimethyl- 170.20
6.668 0.155
Octane, 3-ethyl-2, 7-dimethyl- 170.20
6.768 1.021 Decane, 2, 2, 6-trimethyl- 184.22
6.931 0.360 Undecane 156.19
7.112 0.195 Decane, 3, 3, 5-trimethyl- 184.22
7.173 0.592 Nonane, 3-methyl- 142.17
7.232 0.601 1-Propanol, 2-methyl- 74.07
7.325 0.746 Furan, 4-methyl-2-propyl- 124.09
7.481 0.154 Undecane, 4, 4,-dimethyl- 184.22
7.648 1.807 1-Butanol, 3-methyl-, acetate 130.10
7.836 1.928 2-n-Butyl furan 124.09
8.026 0.279 Benzene, 1 3-dimethyl- 106.08
8.114 0.368 Decane, 3, 3, 5-trimethyl- 184.22
8.303 0.335 Pentane, 1-iodo- 197.99
8.364 1.379 2-Hexanol 102.10
8.498 0.306 Acetic acid, pentyl ester 130.10
8.735 1.228 Hexanoic acid, methyl ester 130.10
9.066 7.956 1-Butanol, 3-methyl- 88.09
9.302 0.134 Phenol, 4-ethyl- 122.07
9.817 0.710 3-Octanone 128.12
10.054 1.780 Acetic acid, hexyl ester 144.12
10.708 0.143 2-Heptanol 116.12
10.985 0.574 7-Octen-2-one 126.10
11.242 6.514 Cyclopropane, propyl- 84.09
11.329 0.550 Acetic acid, sec-octyl ester 172.15
11.545 11.294 Acetic acid, heptyl ester 158.13
11.775 0.205 Octanoic acid, methyl ester 158.13
11.938 0.485 3, 5-Octadiene (Z, Z) 110.11
12.265 3.289 1-Butanol, 3-methyl-, acetate 131.11
The Hydrocarbons of Gliocladium sp.Grown on Oat meal agar 18 days under minimal oxygen
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
14.926 0.254
Cyclopentadiene, 5, 5-dimethyl-1-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
Hydrocarbons ..continued
Gliocladium a virtual chemistry factory
Victoria, Australiatemperate rainforest
The stages of Ascocoryne
The Victoria Fires of 09
SEM’s of Gliocladium sp. and a fossilized fungus
Could fungi have contributed to the formation of crude oil ??
Chief Contributors