instructions for the collection of plant specimens and endophytes studies

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