Antifungal activity of saponins originated from Medicago hybrida against some ornamental plant pathogens

Antifungal activity of total saponins originated from roots of Medicago hybrida (Pourret) Trautv. were evaluated in vitro against six pathogenic fungi and eight individual major saponin glycosides were tested against one of the most susceptible fungi. The total saponins showed fungitoxic effect at all investigated concentrations (0.01%, 0.05% and 0.1%) but their potency was different for individual fungi. The highest saponin concentration (0.1%) was the most effective and the inhibition of Fusarium oxysporum f. sp. callistephi, Botrytis cinerea, Botrytis tulipae, Phoma narcissi, Fusarium oxysporum f. sp. narcissi was 84.4%, 69.9%, 68.6%, 57.2%, 55.0%, respectively. While Fusarium oxysporum Schlecht., a pathogen of Muscari armeniacum, was inhibited by 9.5% only. Eight major saponin glycosides isolated from the total saponins of M. hybrida roots were tested against the mycelium growth of Botrytis tulipae. The mycelium growth of the pathogen was greatly inhibited by hederagenin 3-O-D-glucopyranoside and medicagenic acid 3-O-D-glucopyranoside. Medicagenic acid 3-O-D-glucuronopyranosyl-28-O-D-glucopyranoside and oleanolic acid 3-O-[ -D-glucuronopyranosyl(1 2)-L-galactopyranosyl]-28-O-D-glucopyranoside showed low fungitoxic activity. Medicagenic acid 3-O-a-D-glucopyranosyl28-O-D-glucopyranoside, hederagenin 3-O-[ -L-rhamnopyranosyl(1 2)-D-glucopyranosyl(1 2)-D-glucopyranosyl]28-O-D-glucopyranoside and hederagenin 3-O-D-glucuronopyranosyl-28-O-D-glucopyranoside did not limit or only slightly inhibited growth of the tested pathogen. While 2 , 3 -dihydroxyolean-12 ene-23-al-28-oic acid 3-O-D-glucuronopyranosyl-28-O-D-glucopyranoside slightly stimulated mycelium growth of B. tulipae.


INTRODUCTION
It is commonly known that use of some fungicides can create dangers to health and to our natural environment.Therefore, during the last decade, more and more studies have been carried out on the possibility of using natural plant-based substances, which would be less toxic than those synthetic chemicals.A considerable number of world publications have drawn attention to the possibility of using saponins, as natural fungicides (H o s t e t t m a n n and M a r s t o n, 1955; O l e s z e k, 1996).Searching for new plant originated substances, a couple of years ago, we screened twenty nine Medicago species and found that a few of them were very rich in fungicidal saponins (J u r z y s t a and W a l e r, 1996; J u r z y s t a and B i a ³ y, 1999).Consistently, chemical structure and fungicidal activity of saponins of species such as M. sativa (L e v y et al. 1989, B i a ³ y et al. 1999 Recently, we isolated from roots of Medicago hybrida fourteen triterpene saponins and established their structures (B i a ³ y et al. 2006), but did not study their biological activities.In the present study, antifungal activity of total saponins from roots of this species was evaluated in vitro against six fungi and eight individual major saponin glycosides were tested against Botrytis tulipae, one of the most susceptible fungus.

Total saponins and saponin glycosides
Total saponins and their individual glycosides were isolated from roots of M. hybrida (Pourret.)Trautv.according to the procedure described by B i a ³ y et al. (2006).Shortly, the ground plant material was defatted with chloroform, and then extracted with methanol under reflux.After removal of alcohol, the residue was dissolved in a small volume of water and the solution was placed on a LiChroprep RP 18 column.The column was washed with water and with diluted methanol until colourless solution was obtained.Total saponins were eluted with methanol and dried.Then the obtained total saponins were fractionated on a silica gel column by eluting with n-butanol saturated with water, and individual saponin glycosides were separated from the fractions by means of reversed-phase chromatography on LiChroprep RP-18 columns eluting with aqueous methanol solutions.Saponin structures were established on the basis of hydrolysis and spectral evidence, including IR, optical rotations, NMR and FAB-MS analyses (B i a ³ y et al. 2006).
The total saponins and the following saponin glycosides (Fig. 1) were studied for their antifungal activity: In vitro growth of some pathogenic fungi in the presence of saponins Botrytis cinerea Pers.[Botryotinia fuckeliana (de Bary) Whetzel], Botrytis tulipae (Lib.)Lind, Phoma narcissi Aderh.[syn.Stagonospora curtissi (Berk.)Sacc.],Fusarium oxysporum Schlecht.f. sp.callistephi (Beach.)Snyd.et Hans., Fusarium oxysporum Schlecht.f. sp.narcissi Snyd.et Hans., and Fusarium oxysporum Schlecht., a pathogen of Muscari armeniacumm, were used for investigation.The total amount of saponins originated from roots of Medicago hybrida at final concentrations of 0.01%; 0.05% and 0.1% were previously dissolved in 5 cm 3 distilled and sterilized water and added to potato-dextrose-agar (PDA-Merck) after sterilization at a temperature of about 50 o C. The single saponins isolated from roots of M. hybrida at final concentrations of 10, 25, 50 and 100 mg•cm -3 were dissolved in 3 cm 3 of 75% methanol and added to 100 cm 3 of PDA after sterilization.Five mm diameter plugs taken from 7-day-old culture of tested fungi, were placed in the middle of 90 mm Petri dishes containing PDA medium supplemented with the tested compounds and control without saponins.The plates were incubated at 25 o C in darkness.The diameter of colonies was measured within 4, 5 or 7 days-incubation depending on the fungus being tested.Five dishes were used for each treatment and the experiment was repeated twice.To analyze the differences between mean values, Duncan's test was used, with a significance level of a = 0.05.

RESULTS AND DISCUSSION
The total saponins at concentrations of 0.01%, 0.05% and 0.1% showed strong fungitoxic effect against all of the investigated fungi (Table 1).However, there were variable effects on the growth of those fungi.Saponins greatly inhibited the growth of Fusarium oxysporum f. sp.callistephi, Botrytis cinerea, Botrytis tulipae, Phoma narcissi, Fusarium oxysporum f. sp.narcissi and linear growth of the mycelium of these Concentration in mg•cm -3 fungi, treated with 0.1% solution of saponins, was inhibited by 84.4%, 69.9%, 68.6%, 57.2%, 55.0%, respectively, in relation to the control culture.While, linear growth of the mycelium of Fusarium oxysporum Schlecht., a pathogen of Muscari armeniacum was inhibited only by 9.5%.
It should be mentioned that different concentrations of saponins similarly inhibited mycelium growth of Botrytis cinerea and Botrytis tulipae, or higher concentration slightly less inhibited the mycelium growth of B. cinerea (Table 1).It is suggested that lower concentrations of saponins were sufficient to block all active places (receptors?) in mycelium hyphae.The mechanism of inhibitory effect of saponins on mycelium growth is unknown.It is also possible that higher concentrations of saponins may interact with other endogenous compound(s) of mycelium and finally the inhibitory effect on mycelium growth is slightly decreased.
According to our knowledge, saponins of M. hybrida have never been studied for fungicidal properties, although we have recently shown that saponins of this species possess an insecticidal activity as high as M. arabica or M. murex saponins (S z c z e p a n i k et al. 2004).On the other hand it is known that both of these species are also rich in highly fungicidal saponins (M a r t y n i u k et al.It can be seen that M. hybrida saponins consist of both monodesmosidic and bidesmosidic glycosides (Fig. 1).Looking at the structure of these bidesmosidic glycosides, from the chemical point of view, it is easy to notice that alkaline hydrolysis of most of them could eliminate sugar chains from the ester linkage at C-28 and induce transformation of low biologically active saponins being bidesmosides into high active monodesmoside.Thus, this procedure allows the creation of highly biologically active medicagenic acid 3-O-b-D-glucopyranoside from the saponin 3. Similarly, bidesmosidic saponins 5, 6 and 7 could be converted into monodesmosides: hederagenin 3-O-b-D-glucuronopyranoside, 2b, 3b-dihydroxyolean-12ene-23-al-28-oic acid 3-O-b-D-glucuronopyranoside and medicagenic acid 3-O-b-D-glucuronopyranoside, respectively.Antifungal activity of these hypothetical saponins should be studied but on the basis of data in the literature these saponins are expected to be highly biologically active.
In conclusion, saponins of Medicago hybrida have been shown to possess significant antifungal activity and roots of this plant could be a rich source of natural fungicides.
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; M a r t y n i u k et al. 1996; S a n i e w s k a et al. 2001; 2003), M. arabica (M a r t y n i u k et al. 2004; B i a ³ y et al. 2004; M a r t y n i u k et al. 2004; S a n i e w s k a et al. 2005), M. arborea (T a v a et al. 2005) and M. hybrida (B i a ³ y et al. 2006) have been studied.

Fig. 3 .
Fig. 3. Inhibitory effect of medicagenic acid 3 O b D glucopyranoside (2) in different concentra tion on in vitro mycelium growth of Botrytis tulipae after 2, 4 and 6 days of incubation.