Activity of Haliscosamine against Fusarium oxysporum f.sp. melonis: in vitro and in vivo analysis

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.


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 12 th 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 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. 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 ). This compound is active against human pathogenic yeasts, Candida albicans, Candida tropicalis and Cryptococcus neoformans (El .
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.
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).  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.
Until now, the research that has been conducted on H. viscosa, led to the isolation of a number of alkaloids (Timm et al. 2010 (Schmidt et al. 2012). Two other alkaloids, haliclamine C and D, were isolated from H. viscosa (Volk et al. 2004 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.

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 . 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).

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 10 5 conidia/mL. Each disk 6 mm in diameter received 20 μg of haliscosamine (20 μL of pur haliscosamine at 1 mg/mL in CH 2 Cl 2 [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 DESO-GERME 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 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).