In vitro screening of antifungal activity of marine sponge extracts against five phytopathogenic fungi

The aim of our research is the screening of extracts of marine sponges for their antifungal activity against phytopathogenic fungi. The in vitro screening of hydroalcoholic and organic extracts of ten marine sponges from Atlantic coast of Morocco against five phytopathogenic fungi (Fusarium oxysporum f.sp. melonis, Fusarium oxysporum f.sp. radicis-lycopersici, Fusarium oxysporum f.sp. ciceris, Botrytis cinerea and Penicillium digitatum) showed that only two sponges (Haliclona viscosa and Cynachirella tarentina) are active against all phytopathogenic fungi studied.


Introduction
Agriculture in Morocco is an important economic sector, with 40% of the population living on its revenues. The agricultural area is estimated to be 9.5 million hectares. Fungi are the main responsible agents for losses in agriculture and horticulture and can infect any part of the plant (Messiaen et al. 1991). The fight against these fungi is based on the use of chemical pesticides. However, chemical pesticides sprayed into the air or discharged into the soil can be harmful to the environment and to humans as well.
More than 15 000 natural products were isolated between 1965 and 2005 from marine organisms (Blunt et al. 2007). One of the main factors contributing to this trend is related to modern technology, and ocean biodiversity has become more accessible (Battershill et al. 2005).
Most marine invertebrates, which lack defence structures, have developed chemical defence systems in producing toxic secondary metabolites (Anderson et al. 1994;Aratake et al. 2009).
Sponges consist not only of sponge tissue but also of microorganisms, which represent 50% of their mass. So, is that the antifungal that is secreted by the sponge or a microorganism associated with this sponge? The isolation of microorganisms associated to sponges and screening of antifungal metabolites produced by these microorganisms may provide an answer.
In Morocco, the researches of the metabolism products of medicinal plants and other groups of terrestrial or marine organisms are intensified to explore the possible use of metabolites in different areas. Despite the richness and biodiversity of the Moroccan sea, invertebrates and algae from seabed are poorly studied.
In this study, we report the antifungal activity of ethanol and dichloromethane extracts of ten marine sponges collected from Coastal Atlantic of El Jadida (Morocco) to select the most active species, which could be utilized to purify antifungal compounds.

Results and discussion
The identification of sponge species and their sampling sites are summarized in Table 1.
The results of the screening of antifungal activity of sponge extracts against phytopathogenic fungi are summarized in Table 2.
Organic and hydroalcoholic extracts of H. viscosa exhibit antifungal activity whereas in C. tarentina, only the organic extract is active. This inhibition's effect can be shown in Figure 1.
Up to now, the researches conducted on H. viscosa led to the isolation of a number of alkaloids. Fusetani et al. 1989 (Schmidt et al. 2012). In addition to this, two other alkaloids, haliclamine C and D, were isolated from H. viscosa .
Lately, we have isolated a new product called haliscosamine from H. viscosa (El- ). This product is active against yeasts involved in human pathology. The chemistry of Cinachyrella tarentina sponge is rarely studied (El- Amraoui et al. 2010;El-Wahidi et al. 2011). This sponge was discovered in Italy (Pulitzer-Finali 1983), and now, we have collected this sponge from the Deauville beach, El Jadida, Morocco (El- Amraoui et al. 2010).

Conclusion
Preliminary results have shown that Moroccan sponges constitute a potential source of compounds, which can be used for crop protection. Haliclona viscosa and Cinchyrella tarentina have an interesting antifungal potential. Thus, these sponges provide a potential source of antifungal compound to fight against plant diseases and should be investigated for isolation of this natural compound.

Cliona viridis
The geographical location and depth of sampling sites are shown in Figure 2.

Sponge materials
Ten marine sponges were collected from five sites of the littoral Atlantic coast of El Jadida (Morocco). Figure 2 shows the locations and depths of sampling sponges. All the sponges were identified by Dr. Maria-Jesús Uriz, Research Professor at the Centro de Estudios Avanzados de Blanes (CEAB) and Consejo Superior de Investigaciones Cientificas (CSIC) Spain by morphological characteristics and molecular methods (El- Amraoui et al. 2010). The collected materials were immediately frozen at −4°C for one night prior to extraction.

Preparation of the extracts
Each sponge (100 g wet weight) was thawed, and extracted with ethanol (3 × 100 ml). The ethanol was evaporated at reduced pressure. The suspension was completed with sterile water to 100 ml and extracted with CH 2 Cl 2 (3 × 100 ml).
The CH 2 Cl 2 extract was dried on anhydrous sodium sulphate (Na 2 SO 4 ), then filtered and concentrated at reduced pressure to give a C extract.
The aqueous phase was lyophilised and dissolved twice in absolute ethanol, then filtered and concentrated at reduced pressure to give a B extract (El- Amraoui et al. 2010).

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 Nalkyl dimethyl benzyl ammonium chloride.

In vitro antifungal activity
The antifungal activity was assessed in vitro by agar disc-diffusion test.

Agar disc-diffusion test
This test uses Potato Dextrose Agar (PDA) as medium [Difco]. Conidial suspension was prepared from a 5-dold fungal culture and adjusted with Malassez's cellule in sterile water in order to obtain a final concentration of 10 5 conidia/mL. Each disk received 100 μg of sponge extract (20 μL of each extract at 5 mg/mL were added to each cellulose disc) and was applied on the test media which were previously inoculated with each test strain (El- Amraoui et al. 2010). 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 zones were measured after 24 h of incubation (Galeano and Martınez 2007). Standard disks of DESOGERME SP VEGETAUX® (100 μg) served as positive antifungal control. All the assays were carried out in triplicate.