El Amraoui et al. SpringerPlus (2015) 4:16
DOI 10.1186/s40064-015-0797-x
a SpringerOpen Journal
RESEARCH
Open Access
Activity of Haliscosamine against Fusarium
oxysporum f.sp. melonis: in vitro and in vivo analysis
Belakssem El Amraoui1,2*, Jean François Biard3, Fatima Ez-Zohra Ikbal4, Majida El Wahidi2, Mostafa Kandil5,
Mohammed El Amraoui2 and Aziz Fassouane2,6
Abstract
Marine sponges are a potential source of new molecules with diverse biological activities. We have previously
isolated a sphingosine derivative, (9Z)-2-amino-docos-9-ene-1,3,13,14-tetraol (Haliscosamine) from the Moroccan sea
sponge Haliclona viscosa. The aim of this study was to test Haliscosamine in vitro and in vivo for its antifungal
activity against Fusarium oxysporum f.sp. melonis causing fusarium wilt of melon.
Overall, in vitro test showed that haliscosamine has a similar effect as DESOGERME SP VEGETAUX®. In addition,
in vivo showed a significant effect against Fusarium oxysporum f.sp. melonis. Taking to gather, our results suggest
that haliscosamine constitutes a potential candidate against Fusarium oxysporum f.sp. melonis and the possibility to
use in phytopathology.
Keywords: Fusarium oxysporum; Porifera; Haliscosamine; Marine sponges; Haliclona
Introduction
Agriculture is an important economic sector in Morocco ;
itemploys about 40% of the nation's workforce. The harvest of melon is popular in Morocco; it is found throughout the country. Moreover, Morocco is the 12th largest
exporter to export 55,000 tons of melon in 2009 (El Ouafi
2009). However, diseases that still cause problems in
melon, are especially Fusarium followed by powdery mildew and bacterial blight (Messiaen et al. 1991). Fusarium
wilt caused by Fusarium oxysporum f.sp. melonis (FOM),
is a major disease affecting melon production in the province of El Jadida (Morocco) and causes important economic losses in this area. Thus, the suppression of this
pathogen is considered urgent and a big challenge for this
type of agriculture. Indeed, preventive treatment using
chemical pesticides is the only way to fight these fungi.
However, chemical pesticides sprayed into the air or discharged into the soil can be harmful to the environment
and to humans. Biological antifungal may be an alternative. Furthermore, Marine sponges are a potential source
* Correspondence: elamraouibelkassem@yahoo.fr
1
Faculté Polydisciplinaire de Taroudant, Université Ibn Zohr, Taroudant,
Maroc
2
Laboratoire Contrôle Qualité en Bio-Industrie et Molécules Bio-Actives,
Faculté des Sciences, Université Chouaib Doukkali, BP 20, El Jadida 24000,
Maroc
Full list of author information is available at the end of the article
of new biological compounds with diverse biological activities (Acosta and Rodriguez 1992; Baslow and Turlapaty
1969; Akiyama et al. 2009; Bao et al. 2007a; Bao et al.
2005; Bao et al. 2007b). In Morocco, few studies are carried
out about Moroccan sponges with an important biological
material for the isolation of new molecule (El Amraoui
et al. 2014b; EL Amraoui et al. 2014a; El Amraoui et al.
2013; El Amraoui et al. 2010; El-Wahidi et al. 2011;
El-Wahidi et al. 2013). Haliscosamine isolated from the
Moroccan marine sponge Haliclona viscosa is a new derivative of sphingosine with an original molecular structure
((Z)-2-amino-docos-9- ene-1,3,13,14-tetraol) (El Amraoui
et al. 2013). This compound is active against human pathogenic yeasts, Candida albicans, Candida tropicalis and
Cryptococcus neoformans (El Amraoui et al. 2013).
To put it briefly, the aim of this study was to test the
antifungal activity in vitro and in vivo of haliscosamine
against Fusarium oxysporum f.sp. melonis.
Results and discussion
Antifungal in vitro test has shown that Haliscosamine is
more active than DESOGERME SP against FOM with
inhibition diameters of 21 mm and 19 mm respectively
as illustrated in Figure 1. Haliscosamine showed fungicidal activity against FOM.
© 2015 El Amraoui et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly credited.
El Amraoui et al. SpringerPlus (2015) 4:16
Page 2 of 5
Figure 1 Example of in vitro antifungal activity of Haliscosamine (Ha) and DESOGERME SP (PC) against Fusarium oxysporum f.sp.melonis.
Interestingly, in vivo result showed that the average
number of infected seedlings is significantly lower than the
average number of infected seedlings in the positive control (Figure 2). Fusarium oxysporum f.sp. melonis had been
suppressed by Haliscosamine treatment in infected plant
with different concentrations (1% and 2%). This result indicates that the inhibition of Fusarium was a concentrationdependent manner of Haliscosamine (Figure 2).
Figure 3 shows the percentages of seedlings infected with
the pathogen in each treatment and in the controls. No
seedling (0%) of the negative control uninfected (NCU)
has presented infection while all seedlings (100%) of the
negative control infected (NCI) were infected.
The analysis of variance (ANOVA1) show highly significant (P < 1‰) factor treatment (intergroup variation).
Comparison of means by Duncan's test helped highlight
Figure 2 Average infected seedlings according to the in vivo treatment (NCI: Negative control infested, NCU: Negative control Uninfested,
PC: positive control, Ha: Haliscosamine).
El Amraoui et al. SpringerPlus (2015) 4:16
Page 3 of 5
Figure 3 Percentages of seedlings infected with the pathogen in each treatment and in the controls (NCI: Negative control infested,
NCU: Negative control Uninfested, PC: positive control, Ha: Haliscosamine).
homogeneous groups at the 5%. Haliscosamine 1% has
an effect similar to DESOGERME SP 2% on the inhibition of FOM. In comparison with the positive control,
Haliscosamine has a significant inhibitory effect on the
disease development.
The genus of Haliclona sponges are known for their
high chemical various secondary metabolites with interesting biological activities (Faulkner 2002) including the antifungal (Barrett et al. 1996; Clark et al. 2001; Wattanadilok
et al. 2007), antileishmanial (Dube et al. 2007), antioxidant (Regoli et al. 2004), cytotoxic (Erickson et al. 1997;
Fusetani et al. 1989) and other activities (Hattori et al.
1998; Randazzo et al. 2001; Lakshmi et al. 2009; Roper
et al. 2009).
Until now, the research that has been conducted on
H. viscosa, led to the isolation of a number of alkaloids
(Timm et al. 2010). Fuestani et al. (1989) have isolated
two cytotoxic compounds, Haliclamine A and B from
H. viscosa. Volk and Kock 2003 isolated viscosamine, two
forms of viscosaline have recently been isolated (Schmidt
et al. 2012). Two other alkaloids, haliclamine C and D,
were isolated from H. viscosa (Volk et al. 2004). In a recently published work, Haliclona viscosa has shown
significant antifungal activity against plant pathogenic
fungi from Fusarium, Botrytis and Penicillium genus
(El Amraoui et al. 2014b).
Haliscosamine isolated from Haliclona viscosa sponge,
has a strong antifungal activity with a wide spectrum. It is
active against human pathogenic yeasts (Candida albicans
ATCC 10231, Candida tropicalis R2 CIP 1276.81 and
Cryptococcus neoformans ATCC 11576) (El Amraoui et al.
2013) and against a very resistant phytopathogenic fungus
(Penicilium digitatum) (EL Amraoui et al. 2014a).
Sponges, since a long time have been a major source
of new biomolecules and they are still the inexhaustible
source of new products with different biological activities;
they can be used in various areas. Moroccan sponges are
little studied, and yet they constitute a new biological
material for researchers who are limited to medicinal
plants and beach’s invertebrates and algae.
Conclusion
Haliscosamine isolated from the Moroccan sponge, Haliclona viscosa showed in vitro fungicidal activity against
redoubtable-phytopathogenic fungi. The in vivo studies
of this product against Fusarium wilt showed promising
results. Haliscosamine can be studied more effectively
(open-field activity and toxicity) to see the possibility of
its use as a biopesticide.
Materials and methods
Phytopathogen strains
The phytopathogen strain of the fungus Fusarium oxysporum f.sp. melonis, Fom 20474 CECT (Coleccion Espanola
de Cultivos Tipo) was used in this study (Suárez-Estrella
et al. 2007; Suárez-Estrella et al. 2004).
Haliscosamine
Haliscosamine is an antifungal isolated from the Moroccan
marine sponge Haliclona viscosa. It is a new derivative
of sphingosine with an original molecular structure ((Z)2-amino-docos-9- ene-1,3,13,14-tetraol) and it is active
against human pathogenic yeasts, Candida albicans,
Candida tropicalis and Cryptococcus neoformans. Haliscosamine used in this study, was isolated as described previously (El Amraoui et al. 2013).
DESOGERME SP VEGETAUX®
DESOGERME SP VEGETAUX® (LAKORALE, Morocco),
used in this study as a positive control, is an algaecide,
fungicide and bactericide product used in Morocco both
to remove algae, fungi and bacteria in irrigation systems
and also to disinfect soil. It consists of 20 g/L of polyhexamethyle bioguanidine hydrochlorique and 50 g/L of
N-alkyl dimethyl benzyl ammonium chloride (EL Amraoui
et al. 2014a).
El Amraoui et al. SpringerPlus (2015) 4:16
In vitro antifungal activity
This test uses Potato Dextrose Agar (PDA) as medium
[Difco]. Conidial suspension was prepared from a 5-dold
fungal culture (FOM culture was covered with 10 ml of
distilled water and then scraped with a sterile glass rod;
spores were recovered after filtration on sterile wool cotton) and adjusted with Malassez’s cellule in sterile water
in order to obtain a final concentration of 105 conidia/mL.
Each disk 6 mm in diameter received 20 μg of haliscosamine (20 μL of pur haliscosamine at 1 mg/mL in CH2Cl2
[Difco] were added to each cellulose disc) and then dried
and placed on previously inoculated PDA medium. Plates
were first kept at 4°C for at least two hours to allow the
diffusion of chemicals, and then incubated at 28°C. Inhibition was scored by the absence of any contact between
the discs and fungi after 48 h of incubation then inhibition
zones were measured. Standard disks of the DESOGERME SP VEGETAUX® (20 μl/disc), served as the positive antifungal controls. All the assays were carried out in
triplicate.
To determine whether the haliscosamine has fungistatic
(temporary inhibition) or fungicide (permanent inhibition)
effect on FOM, agar cylinder was cut out from inhibition
zone and placed on the PDA medium and revival of their
growth was observed. The fungicidal effect was where
there was no growth after additional nine days of incubation at 25°C; whereas, a fungistatic effect was where temporary inhibition of mycelial growth occurred (Askarne
et al. 2012).
In vivo antifungal activities of Haliscosamine and
DESOGERME SP VEGETAUX® against Fusarium wilts of
melon
Haliscosamine was assayed in a greenhouse to determine if
it possessed the ability to suppress Fusarium wilt of melon
plants. In these tests, two DESOGERME SP VEGETAUX®
solutions, 1% and 2% were used as positive control.
Initially, seedlings of charentais melon (No resistance
to Fusarium wilt) were planted in 20-cm-diameter pots
containing 2.5 L of sterile substrate [Plantaflor PROFI
TYP3]. The haliscosamine was dissolved in DMSO and
solutions of 1% and 2% were prepared in the irrigation
water. Then, the pots were irrigated for two days by each
of these solutions. On the third day, a 7-d-old FOM culture grown in potato dextrose broth (PDB) was added to
the pots containing plants. Pathogen inoculum which consisted of a mixture of conidia and chlamydospores, was
added to the potting mix at a rate of 1000 propagules/g of
substrate (Suárez-Estrella et al. 2007). Each treatment consisted of five replicate pots of five plants per pot. Disease
was monitored for 6 weeks. Stem sections of all seedlings
were destructively harvested and surface disinfected in
0.5% household bleach (0.0026% sodium hypochlorite)
and placed on PDA to confirm the presence of the
Page 4 of 5
pathogen. Results were shown as the total percentage of
seedlings infected with the pathogen.
Two negative controls (without any prior treatment)
were used:
– Negative control infested (NCI): All plants of NCI
were infested with FOM without any prior treatment.
– Negative control Uninfested (NCU): No plants of
CNU has infected or treated.
Statistical analysis
One-way analysis of variance (ANOVA) was used to highlight the effect of treatment on the development of the
plant pathogen. Averages of infected plants of different
treatments were compared by Duncan test. P-value <0.05
was considered as a significant difference. Statistical analysis of data was performed using the SPSS software package 10.0 (SPSS Inc. USA).
Competing interest
All authors declare that they have no competing interest.
Authors’ contributions
All authors read and approved the final manuscript.
Acknowledgements
We thank Dr. F. Suárez-Estrella from Alméria University, Spain who provide us
with a Fusarium oxysporum f.sp. melonis strains and M. Mouchene, director of
the agricultural department in LACORALE society (Morocco) who provide us
with a DESOGERME SP VEGETAUX® and melon seeds.
Author details
1
Faculté Polydisciplinaire de Taroudant, Université Ibn Zohr, Taroudant,
Maroc. 2Laboratoire Contrôle Qualité en Bio-Industrie et Molécules
Bio-Actives, Faculté des Sciences, Université Chouaib Doukkali, BP 20, El
Jadida 24000, Maroc. 3Laboratoire MMS, Faculté de Pharmacie Université de
Nantes, Nantes, France. 4Laboratoire de Physiologie végétale et
phytopathologie, Faculté des Sciences, Université Chouaib Doukkali, BP 20, El
Jadida 24000, Maroc. 5Laboratoire d’Anthropogénétiques et Biostatistiques,
Faculté des Sciences, Université Chouaib Doukkali, BP 20, El Jadida 24000,
Maroc. 6Ecole Nationale du Commerce et de Gestion ENCG de Settat, Settat,
Maroc.
Received: 31 October 2014 Accepted: 5 January 2015
References
Acosta AL, Rodriguez AD (1992) 11-oxoaerothionin: a cytotoxic antitumor
bromotyrosine-derived alkaloid from the Caribbean marine sponge Aplysina
lacunosa. J Nat Prod 55(7):1007–1012
Akiyama T, Ueoka R, van Soest RW, Matsunaga S (2009) Ceratodictyols, 1-glyceryl
ethers from the red alga-sponge association Ceratodictyon spongiosum/
Haliclona cymaeformis. J Nat Prod 72(8):1552–1554, doi:10.1021/np900355m
Askarne L, Talibi I, Boubaker H, Boudyach EH, Msanda F, Saadi B, Serghini MA, Ait
Ben Aoumar A (2012) In vitro and in vivo antifungal activity of several
Moroccan plants against Penicillium italicum, the causal agent of citrus blue
mold. Crop Prot 40:53–58, doi:10.1016/j.cropro.2012.04.023
Bao B, Sun Q, Yao X, Hong J, Lee CO, Sim CJ, Im KS, Jung JH (2005) Cytotoxic
bisindole alkaloids from a marine sponge Spongosorites sp. J Nat Prod 68
(5):711–715, doi:10.1021/np049577a
Bao B, Sun Q, Yao X, Hong J, Lee CO, Cho HY, Jung JH (2007a) Bisindole alkaloids
of the topsentin and hamacanthin classes from a marine sponge
Spongosorites sp. J Nat Prod 70(1):2–8, doi:10.1021/np060206z
Bao B, Zhang P, Lee Y, Hong J, Lee CO, Jung JH (2007b) Monoindole alkaloids
from a marine sponge Spongosorites sp. Mar Drugs 5(2):31–39
El Amraoui et al. SpringerPlus (2015) 4:16
Barrett AG, Boys ML, Boehm TL (1996) Total Synthesis of (+)-Papuamine: An
Antifungal Pentacyclic Alkaloid from a Marine Sponge, Haliclona sp. J Org
Chem 61(2):685–699, doi:jo951413z
Baslow MH, Turlapaty P (1969) In vivo antitumor activity and other
pharmacological properties of halitoxin obtained from the sponge Haliclona
viridis. Proc West Pharmacol Soc 12:6–8
Clark RJ, Garson MJ, Hooper JN (2001) Antifungal alkyl amino alcohols from the
tropical marine sponge Haliclona n. sp. J Nat Prod 64(12):1568–1571,
doi:np010246x
Dube A, Singh N, Saxena A, Lakshmi V (2007) Antileishmanial potential of a
marine sponge, Haliclona exigua (Kirkpatrick) against experimental visceral
leishmaniasis. Parasitol Res 101(2):317–324
El Amraoui B, Biard JF, Uriz MJ, Rifai S, Fassouane A (2010) Antifungal and
antibacterial activity of Porifera extracts from the Moroccan Atlantic coasts.
J Mycol Med 20(1):70–74, doi:http://dx.doi.org/10.1016/j.mycmed.2009.11.001
El Amraoui B, Biard JF, Fassouane A (2013) Haliscosamine: a new antifungal
sphingosine derivative from the Moroccan marine sponge Haliclona viscosa.
Springerplus 2:252, doi:10.1186/2193-1801-2-252 363
EL Amraoui B, El Wahidi M, Fassouane A (2014a) Control of postharvest green
mould of citrus by haliscosamine isolated from Haliclona viscosa sponge.
EPPO Bulletin 44(1):73–77
El Amraoui B, El Wahidi M, Fassouane A (2014b) In vitro screening of antifungal
activity of marine sponge extracts against five phytopathogenic fungi.
SpringerPlus 3:629, doi:10.1186/2193-1801-3-629
El Ouafi H (2009) Melon: Le Maroc, 12ème exportateur mondial. Agriculture du
Maghreb. Accessed http://www.agriculturedumaghreb.com/agriculture/AdM/
archives/melon.pdf
El-Wahidi M, El-Amraoui B, Biard JF, Uriz MJ, Fassouane A, Bamhaoud T (2011)
Variation saisonnière et géographique de l’activité antifongique des extraits
de deux éponges marines récoltées sur le littoral atlantique d’El Jadida,
Maroc. J Mycol Med 21:28–32
El-Wahidi M, El-Amraoui B, Fassouane A, Bamhaoud T (2013) Isolement bio-dirigé
d’un antifongique à partir de Haliclona enamela récoltée du port de Jorf
Lasfar, Maroc. J de Mycologie Médicale/J Med Mycol 23(2):91–96,
doi:http://dx.doi.org/10.1016/j.mycmed.2013.04.006
Erickson KL, Beutler JA, Cardellina IJ, Boyd MR (1997) Salicylihalamides A and B,
Novel Cytotoxic Macrolides from the Marine Sponge Haliclona sp. J Org
Chem 62(23):8188–8192
Faulkner DJ (2002) Marine natural products. Nat Prod Rep 19(1):1–48
Fusetani N, Yasumuro K, Matsunaga S, Hirota H (1989) Haliclamines A and B,
cytotoxic macrocyclic alkaloids from a sponge of the genus Haliclona.
Tetrahedron Lett 30:6891–6894
Hattori T, Adachi K, Shizuri Y (1998) New ceramide from marine sponge Haliclona
koremella and related compounds as antifouling substances against
macroalgae. J Nat Prod 61(6):823–826
Lakshmi V, Srivastava S, Kumar Mishra S, Misra S, Verma M, Misra-Bhattacharya S
(2009) In vitro and in vivo antifilarial potential of marine sponge, Haliclona
exigua (Kirkpatrick), against human lymphatic filarial parasite Brugia malayi:
antifilarial activity of H. exigua. Parasitol Res 105(5):1295–1301
Messiaen CM, Blancard D, Rouxel F, Lafon R (1991) Les maladies des plantes
maraîchères, 3èmeth edn. INRA, Paris
Randazzo A, Bifulco G, Giannini C, Bucci M, Debitus C, Cirino G, Gomez-Paloma L
(2001) Halipeptins A and B: two novel potent anti-inflammatory cyclic
depsipeptides from the Vanuatu marine sponge Haliclona species. J Am
Chem Soc 123(44):10870–10876
Regoli F, Nigro M, Chierici E, Cerrano C, Schiapparelli S, Totti C, Bavestrello G
(2004) Variations of antioxidant efficiency and presence of endosymbiotic
diatoms in the Antarctic porifera Haliclona dancoi. Mar Environ Res
58(2–5):637–640
Roper KE, Beamish H, Garson MJ, Skilleter GA, Degnan BM (2009) Convergent
antifouling activities of structurally distinct bioactive compounds synthesized
within two sympatric Haliclona demosponges. Mar Biotechnol (NY)
11(2):188–198
Schmidt G, Timm C, Grube A, Volk CA, Kock M (2012) Viscosalines B(1,2) and E
(1,2): challenging new 3-alkyl pyridinium alkaloids from the marine sponge
Haliclona viscosa. Chemistry 18(26):8180–8189, doi:10.1002/chem.201101362
Suárez-Estrella F, Vargas-Garcýa C, Lopeza MJ, Morenoa J (2004) Survival of
Fusarium oxysporum f.sp. melonis on plant waste. Crop Prot 23:127–133
Suárez-Estrella F, Vargas-Garcýa C, Lopeza MJ, Capelb C, Morenoa J (2007)
Antagonistic activity of bacteria and fungi from horticultural compost against
Fusarium oxysporum f.sp. melonis. Crop Prot 26:46–53
View publication stats
Page 5 of 5
Timm C, Mordhorst T, Kock M (2010) Synthesis of 3-alkyl pyridinium alkaloids
from the arctic sponge Haliclona viscosa. Mar Drugs 8(3):483–497,
doi:10.3390/md8030483
Volk CA, Kock M (2003) Viscosamine: the first naturally occurring trimeric 3-alkyl
pyridinium alkaloid. Org Lett 5(20):3567–3569
Volk CA, Lippert H, Lichte E, Köck M (2004) Two New Haliclamines from the
Arctic Sponge Haliclona viscosa. Eur J Org Chem 3154–3158. doi:10.1002/
ejoc.200400026
Wattanadilok R, Sawangwong P, Rodrigues C, Cidade H, Pinto M, Pinto E, Silva A,
Kijjoa A (2007) Antifungal activity evaluation of the constituents of Haliclona
baeri and Haliclona cymaeformis, collected from the Gulf of Thailand. Mar
Drugs 5(2):40–51
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