MARINE BIOPROSPECTING FOR AGRICULTURE

Noer Kasanah,1 David E. Wedge2 and Mark T. Hamann3

1Department of Fisheries, Faculty of Agriculture, Gadjah MadaUniversity, Yogyakarta, Indonesia

2Natural Products Utilization Research Unit, USDA-Agricultural Research Service, USA

3Department of Pharmacognosy, School of Pharmacy, University of Mississippi, USA

INTRODUCTION

� Marine bioprospecting: Mining the untapped potential of living marine resources.

� Oceans harbor myriad organic molecules with utility for the development of pharmaceuticals, neutraceuticals and agrochemicals.

� Objective: Searching for anti fungal agent against Fusarium spp.

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Fusarium spp.

� Common soil saprophyte and plant pathogen

� Problem in agriculture and human health: mycotoxins

� Potential cause of severe opportunistic infections in human immunocompromised patients

� F. solani, F. oxysporium, F. moniliforme, F. verticilloidesand F. proliferatum

� Limited number of antifungal that are effective

� Resistance to the available antifungal agents

Guarro, J and Gene, J. Eur. J. Clin. Microbiol.Infect. Dis. (1995), 14: 741-745.Martino, P., et al. J. of Infections (suppl). 1 (1994), 28: 7-15.

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MARINE NATURAL PRODUCTS

Plakortis sp.

Agelas sp.

4

manzamine A (1), R = H8-hydroxymanzamine A (2), R = OH

NNH

N

N

OHH

HR

ircinal A (3), R = CHO

NH

NOH

N

H

N

HN

N

OH

H

O

OHN

HOO

N

neo-kaulamine

N

N

OH

HR

ircinol A (4), R = CH2OH

4

11

10

9

8

6

1

36

35

34

2620

14

32

23

29

NN

N

N

HH

dehydromanzamine A

NN

N

N

OH

HH R

8-methoxymanzamine A, R = OMe

8-acetoxymanzamine A, R = OAc

NN

N

N

OH

HH

tetrahydromanzamine A, R = Htetrahydro-8-hydroxymanzamine A, R = OH

R

NN

N

N

OH

HH

6-hydroxymanzamine A, R = OH

6-aminomanzamine A, R = NH2

6-methoxymanzamine A, R = OMe

6-n-propoxymanzamine A, R = OnPro

6-(2-ethylhexyloxy)manzamine A, R = O-(2-Et-Hex)

R

NN

N

N

OH

HH

O

OH

manzamine X

NN

N

N

OH

HH

O

OH

manzamine F (5)

Acanthostrongylophora sp.From Manado, Indonesia

Manzamine alkaloids

5

secondaryalcohol

primaryamine

α,β-unsaturated

amide

1

5-MeHex

D-Val-5 L-Thr L-Val-4 D-Val-3

D-Pro L-Orn

D-allo-Ileu-2

D-allo-Thr

D-allo-Ileu-1

D-Val-2

L-Phe

(Z)-Dhb

L-Val-1

O

HN

NH

O HN

O

O

NH

O

NNH

O

H2N

HN

O

O

NH O

O

O

HN O

HN

NH

OO

NHO

HN

HO

Kahalalide F

Elysia rufescens

Bryopsis sp.

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METHODS

� One dimensional bioautography is technique in pre-screening large numbers of extracts, fractions or compounds.

� Microbioassay in a 96-well, micro-dilution broth format was used to evaluate pure compounds identified by bioautography

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One dimensional bioautography

� F. solani, F. proliferatum and F. oxysporium

� 3×105 conidia/mL

� Spray: 0.25% MTT in buffer pH 7

� Standards:

• Amphotericin B

• Captan

• Azoxystrobin

• Benomyl

• Cyprodinil

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Kahalalides-F. proliferatum

F. oxysporum F. proliferatum F. oxysporum

Manzamines-Fusarium

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Microtiter bioassay

� 96-well format

� Determine the sensitivity of fungi to compounds

� Dose-response: 0.3, 3.0 and 30 µM

� Antifungal action: spore germination or mycelialgrowth

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(100%)

(90%)

(80%)

(70%)

(60%)

(50%)

(40%)

(30%)

(20%)

(10%)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

8-OAcMA TH-8-OHMA MA 8-OHMA MF Amp B Azo xys tro bin Captan

Test Samples

% Inhibition -- % Stimulation

0.3 µM 3.0 µM 30 µM

The growth response of F. oxysporum to manzaminealkaloids

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(100%)

(90%)

(80%)

(70%)

(60%)

(50%)

(40%)

(30%)

(20%)

(10%)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

8-OAcMA TH-8-OHMA MA 8-OHMA MF Amp B Azo xys tro bin Captan

Test Samples

% Inhibition -- % Stimulation

0.3 µM 3.0 µM 30 µM

The growth response response of F. proliferatum to manzamine alkaloids

12

(100%)

(90%)(80%)

(70%)

(60%)

(50%)(40%)

(30%)

(20%)(10%)

0%

10%20%

30%

40%50%

60%

70%

80%90%

100%

8-OAcMA TH-8-OHMA MA 8-OHMA MF Amp B Azoxystrobin Captan

Test Samples

% Inhibition -- % Stimulation

0.3 µM 3.0 µM 30 µM

The growth response response of F. solani to manzamine alkaloids

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MECHANISMS OF ANTIFUNGAL RESISTANCE

Antifungal resistance:

Failure of fungal infection to respond to antifungal

Types of resistance:

� Primary resistance (Intrinsic resistance)

Zygomycetes: Rhizopus arrhius, Scedosporium spp., Acremonium spp., Fusarium spp.

� Secondary resistance

Kontoyiannis, D and Lewis, R.E. 2002. The Lancet. 359: 1135-44

Perea, S and Patterson, T.F. 2002. Clin. Infect. Dis. 35: 1073-80.

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MECHANISMS OF ANTIFUNGAL RESISTANCE

� Alteration of target

� Alteration of enzyme (biosynthesis or metabolism)

� Efflux pumps

� Decrease in drug accumulation

� Modification or degradation of compound

White, T.C., et al. 1998. Clin. Microbiol. Rev. 11: 382-402

Loeffler, J and Stevens, D.A. 2003. Clin. Infect. Dis. 36 (Suppl): S31-S41

Morrissey, J.P and Osbourn, A.E. 1999.Microbiol. Mol. Biol. Rev. 63: 708-724.

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Biodegradation of manzamine alkaoids16

Kahalalide F and its derivates

F. proliferatum Growth Response to Samples at 72 hrs.

(100%)(90%)(80%)(70%)(60%)(50%)(40%)(30%)(20%)(10%)

0%10%20%30%40%50%60%70%80%90%

100%

KF (1) 5 7 16 4 8 3 10 Amp B Capt Azoxy

Test Samples

% Inhibition -- % Stimulation

0.3 µM

3.0 µM

30 µM

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F. solani Growth Response to Samples at 72 hrs.

(100%)(90%)(80%)(70%)(60%)(50%)(40%)(30%)(20%)(10%)

0%10%20%30%40%50%60%70%80%90%

100%

KF (1) 5 7 16 4 8 3 10 Amp B Capt Azoxy

Test Samples

% Inhibition -- % Stimulation

0.3 µM

3.0 µM

30 µM

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F. proliferatum Growth Response to Samples at 72 hrs.

(100%)(90%)(80%)(70%)(60%)(50%)(40%)(30%)(20%)(10%)

0%10%20%30%40%50%60%70%80%90%

100%

KF (1) 5 7 16 4 8 3 10 Amp B Capt Azoxy

Test Samples

% Inhibition -- % Stimulation

0.3 µM

3.0 µM

30 µM

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Growing hyphae and mycelia of F.

oxysporum in medium PDB without KF.

The hyphae of F. oxysporum exposed to KF at

concentration 5 µM after germination

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The hyphae of F. proliferatum exposed to KF

at concentration 5 µM after germination

Twisted and destruction hyphae of F.

oxysporum due to long exposure of KF

(after 7 days)

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Mycelia of F. proliferatum after 3 days exposure with fluorescent

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CONCLUSIONS

� Sceptrin, ilimaquinon and Plakortides are inactive against Fusarium spp.

� Fusarium spp are resistance to manzamina alkaloid through mechanism modification or degardation of compounds

� Kahalalide F is active against Fusarium spp, interfere growing hyphae and damage the mycelia

� Marine bioprospecting for new agrochemical compounds is complicated by the nature of compounds: complex structure, low yield, physicochemical properties

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Acknowledgments

� Dr. Abbas S. Gholipour

� Dr. Anna Kochanowska

� Dr. Rabab Muhammad

� Lorei Lucas, Pharm.D

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THANK YOU

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