THE EFFECT OF MEDICAGO ARABICA , M . HYBRIDA AND M . SATIVA SAPONINS ON THE GROWTH AND DEVELOPMENT OF FUSARIUM OXYSPORUM SCHLECHT F . SP . TULIPAE APT

In the present work it was shown that total saponins originated from M. hybrida and M. sativa substantially limited mycelium growth of F. oxysporum f. sp. tulipae and symptoms of fusariosis on tulip bulbs. Out of 15 individual tested saponins originated from M. arabica, M. hybrida and M. sativa, four compounds: 3-O-[β-D-glucopyranosyl (1→2)α-L-arabinopyranosyl] hederagenin, hederagenin 3-O-β-D-glucopyranoside, medicagenic acid, medicagenic acid 3-O-β-D-glucopyranoside had the strongest inhibitory effect on mycelium growth of Fusarium oxysporum f. sp. tulipae on PDA medium. The total saponins from M. arabica, M. hybrida and M. sativa inhibited the number of colony forming units of Fusarium oxysporum f. sp. tulipae in artifi cially infested substrate. The use of saponins originated from Medicago as a fungicide is suggested.

sativa, the following four saponins greatly inhibited linear mycelium growth of Botrytis tulipae and Phoma narcissi: 1→2)-α-L-arabinopyranoside] medicagenate and medicagenic acid (Saniewska et al. 2003).Total saponins originated from shoots of Medicago arabica showed antifungal activity in vitro against the following 11 tested taxons of pathogenic fungi: Alternaria alternata, Botrytis cinerea, Botrytis tulipae, Pestalotia spp, Phoma narcissi, Phoma poolensis, Pythium ultimum, Rhizoctonia solani, Fusarium oxysporum f. sp.callistephi, Fusarium oxysporum f. sp.tulipae, Fusarium oxysporum f. sp.narcissi (S a n i e w s k a et al. 2005).The total saponins of Medicago arabica used preventively to treat tulip bulbs inhibited the development of Fusarium oxysporum f. sp.tulipae (S a n i e w s k a et al. 2005).Total saponins originated from the roots of Medicago hybrida substantially inhibited mycelium growth of Botrytis cinerea, Botrytis tulipae, Fusarium oxysporum f. sp.callistephi, Fusarium oxysporum f. sp.narcissi, Phoma narcissi (S a n i e w s k a et al. 2006).Eight individual major saponin glycosides isolated from M. hybrida roots were tested for their effect on mycelium growth of Botrytis tulipae and it was documented that hederagenin 3-O-β-D-glucopyranoside and medicagenic acid 3-O-β-D-glucopyranoside greatly inhibited the growth of the pathogen (S a n i e w s k a et al. 2006).
In the present study, the effect of total and individual saponins from Medicago arabica, M. hybrida, and M. sativa on growth and development of Fusarium oxysporum f. sp.tulipae is evaluated in vitro and in vivo.

MATERIALS AND METHODS
The study was conducted in 2005-2006 at the Research Institute of Pomology and Floriculture in Skierniewice.Four isolates of Fusarium oxysporum f. sp.tulipae, strongly pathogenic to tulip bulbs, were used.  . 1999), according to the procedures described in the papers.Shortly afterwards, the ground plant material was defatted with chloroform, and then extracted with methanol under refl ux.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 a 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.

Isolation
The total saponins and the following saponin glycosides isolated from M. arabica, M. hybrida and M. sativa were studied from the point of view of their effect on growth and development of Fusarium oxysporum f. sp.tulipae: In vitro growth of Fusarium oxysporum f. sp.tulipae in the presence of saponins.Four isolates of Fusarium oxysporum f. sp.tulipae (F.ox.t. 17, F.ox. t. 36, F.ox.t. 188 and F.ox.t. 218) were used for the investigations.The total amount of saponins, isolated from Medicago hybrida and M. sativa at fi nal concentrations of 0.01%; 0.05%, 0.1%, was previously dissolved in 5 cm 3 sterile distilled water and added to potato-dextrose-agar (PDA-Merck) after sterilization at about 50 o C. Single saponins isolated from of Medicago arabica, M. hybrida and M. sativa at fi nal concentrations of 8 -140 μg cmֿ³ were dissolved in 3 cm 3 of 75% methanol and added to 100 cm 3 of PDA after sterilization.5-mm diameter plugs, taken from 7-day-old culture of the tested isolates, were placed in the middle of 90 mm Petri dishes containing PDA medium supplemented with the tested compounds and the control without saponins.The plates were incubated at 25ºC in darkness.The diameter of the colonies was measured after 4, 6 and 8 days of incubation, depending on the fungus tested.Five dishes were used for each treatment and the experiment was repeated twice.To analyse the differences between mean values, Duncan's test was used, with a signifi cance level of α = 0.05.
The infl uence of saponins on the colony forming units (cfu) of Fusarium oxysporum f. sp.tulipae.Peat-sand (2:1) was artifi cially infested with F. oxysporum f. sp.tulipae (F.ox.t.218) using the procedures of T r a m i e r et al. (1983) and O r l i k o w s k i and S a n i e w s k a (1988).After two-week incubation of inoculated substrate at 25ºC in darkness, twenty pots (100 cm 3 ) were fi lled with 60 g of inoculated substratum samples (4 x 5 reps).Then, water suspension of total saponins from the used species of Medicago at a concentration 5.0% was added to the inoculated substrate (three times every 7 days).After 7, 14 and 21 days of incubation of the substratum inoculated and treated with saponins, the population of the pathogen was determined using K o m a d a (1975) medium.The inoculated substratum watered only without saponins served as the control.From each sample, 10 g of soil was taken, diluted with 90 cm 3 of sterile distilled water and mixed in a magnetic blender for 30 minutes.One millilitre of the suspension was mixed with 50 cm 3 of K o m a d a (1975) medium at a temperature of 40 -45ºC and poured into 90 mm Petri dishes.After a 3day-long incubation of the plates at 25ºC in the dark and 2-day-long incubation in daylight, the number of F. oxysporum f. sp.tulipae colonies was counted as mean values from ten Petri dishes (ø 90 mm), and population densities were converted to the number of propagules per gram of air dry soil.The experiment was repeated twice.Duncan's multiple range t-test at 5% level of signifi cance was used for mean separation.
The effect of total saponins originated from Medicago sativa and M. hybrida on the development of fusariosis on tulip bulbs.'Apeldoorn' tulip bulbs at the fl ower bud stage, uncooled, after removal of the fi rst outer scale, were surface disinfected in 50% ethanol for 5 min., and then rinsed 3 times in sterile distilled water.Then, the bulbs were cut at the basal plate with a cork borer to a depth 1-2 mm and at a diameter of 3 mm.After cutting the scale, the bulbs were soaked for 60 min.in saponin solutions at concentrations of 0.1% and 0.5%.Thereafter, 20 μl of spore suspension of F.ox.t.218 with a density of 1.4 x 10 6 spores cm 3 of the inoculum was put in the place of the bulb scale damage.The bulbs soaked for 60 min.in distilled water and, inoculated with the suspension of the pathogen spores, were the control.The inoculated bulbs were placed in trays on damp absorbent paper lined with aluminium foil.The trays with the bulbs were put into fi lm bags in order to increase humidity.After 10 days of incubation of the bulbs at a temperature of 25ºC, the length of infection spots was measured.
The comparative fungicide was Sportak Alpha 380 EC (300 g prochloraz + 80 g carbendazim dm 3 ).In each combination, 10 bulbs were tested.The experiments were carried out in two series on the bulbs obtained in the study years.

RESULTS AND DISCUSSION
In vitro growth of Fusarium oxysporum f. sp.tulipae in the presence of saponins The total amount of saponins originated from the roots of M. hybrida and of alfalfa (M.sativa) at concentrations of 0.01%, 0.05% and 0.1% showed strong fungitoxic effect against F. oxysporum f. sp.tulipae (F.ox.t.).However, there were variable effects on the growth of all the studied isolates.The growth inhibition of F.ox.t.isolates was generally proportional to the concentration of saponins.The linear growth of the mycelium of the tested pathogen isolates, treated with a 0.1% solution of saponins isolated from M. hybrida and M. sativa was inhibited in 73% -80% and 46% -69%, respectively, in relation to the control culture (Figs 1, 2).In earlier studies (Saniewska et al. 2005), it was documented that total saponins from M. arabica at a concentration of 0.1% limited the mycelium growth of F.ox.t. 17 in vitro at 63%.
The individual saponin glucosides exerted different effects on the mycelium growth of the isolates of Fusarium oxysporum f. sp.tulipae.14) 2β,3β-dihydroxyolean-12ene-23-al-28-oic acid 3-O-β-D-glucuronopyranosyl-28-O-β-D-glucopyranoside did not limit but stimulated the growth of the tested pathogen (Table 1).( 6 slightly stimulated mycelium growth of F. oxysporum f. sp.tulipae at low concentrations and showed slight fungitoxic activity at higher concentrations (Tab.1).The individual saponins from Medicago which greatly inhibited mycelium growth of F. oxysporum f. sp.tulipae were also most active against other pathogens of ornamental plants (S a n i e w s k a et al. 2003, 2006).The infl uence of saponins on colony forming units (cfu) of Fusarium oxysporum f. sp.tulipae The total saponins from M. arabica, M. hybrida and M. sativa decreased the number of colony forming units (cfu) of Fusarium oxysporum f. sp.tulipae in artifi cially infested substrate.However, there were variable effects on cfu of the tested saponins of different species of Medicago.The total saponins used at a concentration of 5.0% reduced the number of cfu of the pathogen of M. sativa in 92.5%, M. arabica in 60% and M. hybrida in 40%, in comparison to the control substrate without saponins (Tab.2).It is the fi rst report that saponins of Medicago decrease the number of colony forming units of Fusarium oxysporum f. sp.tulipae in substrate.

The effect of total saponins from Medicago sativa and M. hybrida on the development of fusariosis on tulip bulbs
Total saponins from M. hybrida and M. sativa used preventively to treat tulip bulbs, inhibited the development of F. oxysporum f. sp.tulipae (Tab.3).The size of necrosis on the surface of scale of bulbs was smaller, being totally inhibited at a concentration of 0.1%, like in the case of prochloraz + carbendazim (Sportak Alpha 380 EC).Disease development was not observed in internal tissues or with a minimum increase during the incubation of the bulbs in comparison with the control bulbs which were not treated.The control bulbs showed the development of the mycelium and gummosis in the area of inoculation.Such sings of development of the mycelium of the pathogen were not observed on the bulbs treated with the saponis solution.Similar results, concerning the limitation of disease symptoms caused by Fusarium oxysporum f. sp.tulipae on tulip bulbs, were documented earlier for saponins isolated from Medicago arabica (S a n i e w s k a et al. 2005).It is suggested that saponins of these three species of Medicago which showed considerable fungitoxic activity against Fusarium oxysporum f. sp.tulipae may be used as a natural fungicide for limiting the spread of the pathogen on tulip bulbs.
of total saponins and saponin glucosides Total saponins and their individual glucosides were obtained from the aerial parts of Medicago arabica L. (B i a ł y et al. 2004), and from the roots M. of hybrida (Pourret) Trautv.(B i a ł y et al. 2006) and M. sativa L. (B i a ł y et al

Table 1
The effect of individual saponins indicated in Material and Methods as number 1

Table 2
Infl uence of saponin solutions on colony forming units (cfu) of F. oxysporum f. sp.tulipae.

Table 3
Effect of preventively used saponins on the development of fusariosis on tulip bulbs cv.Apeldoorn.
SeeTable 2 Fig. 1.Inhibitory effect of total saponins originated from roots of Medicago hybrida on in vitro mycelium growth of four isolates of Fusarium oxysporum f. sp.tulipae (F.ox.t.17, F.ox.t.36, F. ox.t.188, F.ox.t.218) after eight days of incubation; means followed by the same letters are not signifi cantly different at P=0.05 according to Duncan's test.Fig. 2. Inhibitory effect of total saponins originated from roots of Medicago sativa on in vitro mycelium growth of four isolates of Fusarium oxysporum f. sp.tulipae (F.ox.t.17, F.ox.t.36, F. ox.t.188, F.ox.t.218) after eight days of incubation; means followed by the same letters for each isolate are not signifi cantly different at P=0.05 according to Duncan's test.