Control of plant parasitic nematodes with active saponinsand biomass from Medicago sativa

T. D’Addabbo • T. Carbonara • P. Leonetti •

V. Radicci • A. Tava • P. Avato

Received: 29 September 2009 / Accepted: 12 April 2010

� Springer Science+Business Media B.V. 2010

Abstract Medicago sativa L., alfalfa, is the most

known plant species within the Medicago genus. The

plant has been extensively studied for its content of

saponins, mainly consisting of triterpene glycosides

of medicagenic acid, possessing several biological

properties including a biocidal activity on different

soil microorganisms. Phytoparasitic nematodes are

responsible for heavy economic damages to numer-

ous agricultural crops and, due to their large distri-

bution, they are among the most difficult crop pests to

control. Attention on environmental safety and

human and animal health has led to the progressive

dismission of many synthetic formulations for the

control of those pests and to the search of alternative

strategies, including the use of natural metabolites

from plants. Saponins from M. sativa may be good

candidates for natural nematicide formulations, as in

our in vitro studies the saponin mixtures from

M. sativa tissues have been found effective in vitro

against the virus-vector nematode Xiphinema index,

the root-knot nematode Meloidogyne incognita and

the potato cyst parasite, Globodera rostochiensis. A

structure–activity relationship among saponins and

related prosapogenins and sapogenin, respectively,

has also been analyzed. The nematicidal efficacy

differed among the three assayed nematode species,

G. rostochiensis being the most susceptible to the

active compounds from alfalfa. The in vitro results

were also confirmed by experiments in potting mixes

infested by M. incognita or G. rostochiensis and

amended with dry top and root material from

M. sativa, and in field trials on M. incognita and

carrot cyst nematode Heterodera carotae with

M. sativa pelleted meal. All amendments reduced

root and soil population densities of target nematode

species compared to non-treated and chemical con-

trols, with a general improvement of plant growth and

yield performances.

Keywords Alfalfa � Biocide � Medicagenic acid �Phytoparasite nematodes � Sapogenins

Introduction

The genus Medicago (Fabaceae, Faboideae) includes

83 different species, the most known represented by

M. sativa L. (syn. M. media Pers.), commonly named

alfalfa or lucerne (Heyn 1963; Lesins and Lesins

1979; Small and Jomphe 1989). The plant is a

T. D’Addabbo � P. Leonetti � V. Radicci

Istituto per la Protezione delle Piante, CNR,

Via Amendola 165/a, 70126 Bari, Italy

T. Carbonara � P. Avato (&)

Dipartimento Farmaco–Chimico, Universita di Bari,

Via Orabona 4, 70125 Bari, Italy

e-mail: avato@farmchim.uniba.it

A. Tava

C.R.A–FLC Centro di Ricerca per le Produzioni

Foraggere e Lattiero-Casearie, V.le Piacenza 29,

26900 Lodi, Italy

123

Phytochem Rev

DOI 10.1007/s11101-010-9180-2

969

perennial herb, native to Asia but considerably

adaptable to a large range of environments and now

worldwide cultivated as a highly valuable forage

crop.

The genus Medicago contains a variety of chem-

ical classes of secondary metabolites such as couma-

rins, isoflavones, naphtoquinones, alkaloids and

saponins (Barnes et al. 2002). The chemical structure

of saponins from the different species has been

especially studied (Timbekova and Abubakirov

1986a, b; Massiot et al. 1988, 1991; Oleszek et al.

1988; Bialy et al. 1999, 2004, 2006; Huhman and

Sumner 2002; Huhman et al. 2005; Kapusta et al.

2005a, b; Tava et al. 2005, 2009; Tava and Avato

2006); they generally consist of a complex mixture of

triterpene glycosides with medicagenic acid, heder-

agenin, zanhic acid, bayogenin and soyasapogenols A

and B as the dominant aglycones (Tava and Avato

2006). Recently, new aglycone moieties have been

identified from M. arborea (Tava et al. 2005),

M. hybrida (Bialy et al. 2006) and M. arabica (Tava

et al. 2009).

Due to their chemical, physical and physiological

characteristics, naturally occurring saponins display a

broad spectrum of biological and pharmacological

effects (Price et al. 1987; Cheeke 1996; Jurzysta and

Waller 1996; Oleszek 1996; Tava and Odoardi 1996;

Lacaille-Dubois and Wagner 2000; Oleszek 2000;

Sprag et al. 2004; Tava and Avato 2006). They are

distinguished by their surfactant and hemolytic

properties, but they are also reported to possess

fungicidal, molluscicidal, antibacterial, antiviral and

antitumor activities (Tava and Avato 2006).

Cytotoxic effect of saponins from other plants is

reported (Messina et al. 1994; Rao and Sung 1995;

Bader et al. 1996; Huang et al. 2008; Kuljanabhaga-

vad and Wink 2009), but cytotoxicity and antitumor

activity of saponins from Medicago species have not

been investigated in great details (Tava and Odoardi

1996; Tava and Avato 2006; Avato et al. 2008).

Preliminary in vitro assays with purified saponins

from different plant tissues of M. arabica, M. arborea

and M. sativa on HeLa and MCF-7 (cisplatin

resistent) tumor cell lines showed that saponins from

M. arabica were the most active with a toxicity

comparable to that of cisplatin at 100 and 200 lg/mL,

especially against HeLa cell lines (*80%) and their

toxicity was in general increased in combination with

cisplatin (1 and 10 lM).

In contrast, the antibiotic effects of Medicago

saponins have been assessed by several studies

(Jurzysta and Waller 1996; Oleszek 1996; Zehavi

and Polacheck 1996; Levy et al. 1989; Gestetner et al.

1971; Jurzysta and Bialy 1999; Oleszek 2000; Tava

and Avato 2006). Dry meals, saponin extracts and

purified saponins from different Medicago species

have been found effective in vitro against human

pathogenic fungi and bacteria (Polacheck et al. 1986;

Evron et al. 1988; Houghton et al. 2006; Avato et al.

2006) as well as against phytopatogenic fungi

(Martyniuk et al. 1995a, b; Zehavi and Polacheck

1996; Saniewska et al. 2001; Saniewska et al. 2003;

Tava and Avato 2006). Investigations on the struc-

ture–activity relationship of those saponins proved

that in general bioactivity depends on the involved

genin and number of sugars in the molecules (Tava

and Avato 2006).

In vitro physiological activity of saponins from

M. sativa on soil and rhizosphere bacteria has also

been investigated and results obtained suggested that

their efficacy is in most cases due to the aglycone,

medicagenic acid (Hoagland et al. 2001).

The allelopathic effect of M. sativa and its

saponins has been reported in several papers (Macias

et al. 2008). Mishustin and Naumova (1955) observed

that growth of cotton was influenced by the use of

alfalfa as rotation crop. Later investigations (Waller

1989; Guenzi et al. 1964; Waller et al. 1993, 1995)

have shown that, depending on their concentration,

medicagenic acid glycosides may function as plant

growth inhibitors (high concentrations) or stimulators

(low concentrations). Comparison of the allelopathic

effects of structurally different saponins from Medi-

cago species revealed that, as for their antimicrobial

activity, monodesmosides are in general more active

than the related bidesmosides (Tava and Avato 2006).

Phytoparasitic nematodes are distributed world-

wide and are responsible for heavy economic dam-

ages to numerous crops of agriculture interest. They

feed on living plant tissues, either invading the plant

root system and feeding on the root cortex cells

(endoparasites) or living in the soil and feeding on the

rhizodermis cells (ectoparasites).

In particular, the root-knot nematode species

Meloidogyne incognita (Kofoid & White) Chitwood

shows an outstanding diversity of host plants and is

recognized as one of the most destructive species

(Lamberti and Taylor 1979). The golden cyst

Phytochem Rev

123970

nematode Globodera rostochiensis Wollenweber

characterized by the formation of pronounced yel-

lowish cysts on the plant roots, is spread in almost all

potato-growing areas (Behrens 1975; Brown 1969),

whereas the carrot cyst nematode Heterodera carotae

Jones may cause considerable yield losses to carrot

crops in temperate areas (Greco et al. 1993). The

ectoparasite dagger nematode Xiphinema index

Thorne & Allen is considered the most harmful

nematode species to grapevine (Vitis spp.), mainly as

vector of the grapevine fanleaf virus (Brown et al.

1995).

Control of these plant parasitic nematodes has

always been dependent on synthetic nematicides.

Nevertheless the ongoing evolution and application

of government Regulations (Uri 1998; Hynes and

Boyetchko 2006; Reg. CE 396/2005; 1095/2007 and

33/2008) to mitigate the adverse effects of the use of

chemical pesticides on environment and animal and

human health has led to the dismission of most

synthetic nematicides and the development of alter-

native control strategies (Ghorbani et al. 2008).

Plant derived metabolites have attracted most of

the attention as possible alternative pesticides (Duke

1990; Chitwood 2002), either through the use of plant

extracts and phytochemical formulations or as

organic amendments to soil (Akhtar and Malik 2000).

Numerous plant species have been reported to

contain metabolites with nematicidal activity (Bilg-

rami 1997; Gonzalez and Estevez-Braun 1997; Bar-

Eyal et al. 2006; Kong et al. 2006; Batish et al. 2008;

Shakil et al. 2008) and there is a continuous effort in

discovering new plant sources for nematicidal

compounds.

The most known are probably the Brassicaceae

synthesizing the active glucosinolates which, accord-

ing to their chemical structure, decompose in the soil

to release a variety of isothiocyanate derivatives with

nematotoxic action (Zasada and Ferris 2004; D’Add-

abbo et al. 2007).

Another well recognized example of plant effec-

tive to control phytoparasite nematodes is represented

by neem, Azadirachta indica, containing typical

tetranortriterpenoids such as azadirachtin (Akhtar

2000; Raizada et al. 2001; Javed et al. 2008).

Few data are available on the activity of saponins

against plant parasitic nematodes and, to the best of our

knowledge, there is no information on their mecha-

nism of action. Reports are mainly related to the assay

of Quillaja saponaria Molina preparations (Pelah et al.

2002; San Martin 2004; D’Addabbo et al. 2005; San

Martin and Magnunacelaya 2005). Aqueous extracts

from this plant containing a mixture of saponins (25%)

and polyphenols (6%) resulted in a satisfactory

nematode control even at low dosage (San Martin

and Magnunacelaya 2005), associated also to an

increase of crop yield (D’Addabbo et al. 2005).

Saponins from M. sativa, as showing a well

characterized chemical composition and well estab-

lished biological activities, seem to represent good

candidates for phytonematode control. Thus, in the

recent years our interest has been focused on the

study of the nematicidal potential of Medicago and its

active metabolites.

Results from our in vitro experiments on the

biocidal activity of M. sativa saponins on X. index,

M. incognita and G. rostochiensis and from trials in

potting mixes and in field on the efficacy of soil

amendments with alfalfa biomass for the manage-

ment of M. incognita, G. rostochiensis and H. carotae

will be reviewed in this paper. Data from in vitro

assays on G. rostochiensis and partly on M. incognita

are reported here for the first time.

Chemical profile of saponins in M. sativa

Saponins are a large group of glycosidic secondary

metabolites produced by many plant species includ-

ing major food crops. Three major chemical classes

are found: steroid glycosides; steroid alkaloid glyco-

sides and triterpene glycosides, which include the

largest number of structures. They may have one

(monodesmosidic) or more sugar chains (bi-, trides-

mosidic), linear or branched, linked to the aglycone

mojety (sapogenin) through an ether or ester bond.

Their distribution in plants seems to be correlated

to the structural type, that is steroidal saponins are

almost exclusively present in Monocot species, while

triterpenoid saponins mainly occur in the Dicotyle-

dones (Bruneton 1999; Sprag et al. 2004).

Saponins found in the genus Medicago are triter-

pene glycosides and include different structural types,

distinguished by their aglycones and sugars, which

have some chemotaxonomic relevance to discrimi-

nate among the various species within the genus

(Oleszek et al. 1988; Massiot et al. 1991; Oleszek

1996, 2000; Bialy et al. 2004, 2006; Huhman et al.

Phytochem Rev

123971

2005; Kapusta et al. 2005a, b; Tava et al. 2005, 2009;

Tava and Avato 2006).

Although the exact function of saponins in Medi-

cago plants is not fully understood, they are regarded

as constitutive resistance factors involved in defense

mechanisms especially against pathogens. Avenacin

from oat, and tomatine from tomato, for examples,

are known saponins constitutively produced in order

to prevent pathogen attacks.

Saponins from M. sativa have been the subject of

many detailed chemical studies (Massiot et al. 1988,

1992; Oleszek et al. 1990, 1992a, b; Bialy et al. 1999;

Huhman and Sumner 2002; Tava and Avato 2006)

and a summary of the main structures is described in

Table 1. They consist of different glycosilated trite-

rpenic sapogenins (aglycone moieties) such as med-

icagenic acid, zanhic acid, hederagenin, bayogenin

and soyasapogenols A, B, and E. Medicagenic acid

represents far the dominant sapogenin, accounting for

40–70% of the total aglycones depending on the plant

tissue (Tava et al. 1993, 1999; Tava and Pecetti 1988;

Oleszek 1998; Pecetti et al. 2006).

Monodesmosidic saponins, with a sugar chain

linked at the C-3 position of the aglycone, as well as

bidesmosidic saponins, with an additional sugar chain

at the C-28 position have been isolated from this

species (Tava and Avato 2006). A tridesmoside

saponin, with an extra sugar at the C-23 position

has also been reported (Oleszek et al. 1992b). The

most abundant sugars in the alfalfa saponins are the

monosaccharide units glucose, arabinose, rhamnose,

xylose and glucuronic acid.

Variations in the aglycones and their combinations

with sugar chains cause the saponins from M. sativa

to form complex mixtures of different glycosides

which can, as a whole, influence their biological

properties.

A few unusual acylated saponins (Table 1) have

been isolated from the species such as two malonated

saponins (Huhman and Sumner 2002) and one

methylated saponin from the plant roots (Bialy

et al. 1999) and a maltol coniugate from the seeds

(Massiot et al. 1992). It is still questioning however if

they are true metabolites or artifacts produced during

the extraction procedures (Tava et al. 2003).

Detailed studies with alfalfa, have shown that

content and compositional profile of saponins is

influenced by several factors such as environment,

genotype, physiological stage of growth, and plant

part (Pedersen et al. 1967; Pedersen and Wang 1971;

Berrang et al. 1974; Quazi 1975; Tava et al. 1993,

1999; Tava and Pecetti 1998; Pecetti et al. 2006).

Saponins from M. sativa aerial parts (tops) are

characterized by high amounts of medicagenic acid

glycosides (40–60%), followed by zanhic acid (10–

25%), hederagenin (2–8%), bayogenin (3–7%) and

soyasapogenol (10–20%) glycosides. Saponins from

M. sativa roots contain instead higher amount of

medicagenic acid (50–70%), hederagenin (3–10%)

and bayogenin (3–10%) and less amount of soyas-

apogenols (2–5%) and zanhic acid (\1%) (Tava et al.

1993, 1999; Tava and Pecetti 1998; Pecetti et al.

2006). Moreover, saponins from the aerial part are

mainly bidesmosidic saponins, while monodesmoside

saponins are more abundant in the roots (Tava and

Avato 2006).

Significant differences in the chemical composition

of saponins have been found among cutting dates

(Pecetti et al. 2006), especially in the relative amounts

of medicagenic and zhanic acid, which seem to

accumulate preferentially during the earlier stages of

the plant development (Tava and Avato 2006).

Combining all the results from the extensive

chemical analysis of M. sativa, as well as of all the

other Medicago species, appears clear that the

chemical characterization of the saponin extracts

and/or of the different plant tissues used in the assay

is a prerequisite for the reliable understanding of the

biological effects.

Nematicidal activity of saponins from M. sativa

The in vitro biocidal effects of saponin mixtures from

alfalfa top and root tissues were investigated against

the three nematode species X. index (Argentieri et al.

2008), M. incognita (D’Addabbo et al. 2009) and

G. rostochiensis.

Saponin extracts from M. sativa tops used in these

assays contained 52% medicagenic acid, 8% zanhic

acid, 4%, hederagenin, 2% bayogenin, whereas the

saponin root extract contained 65% medicagenic

acid, 3% hederagenin, \1% zanhic acid. Second-

stage juveniles of M. incognita or G. rostochiensis

and adult females of X. index were exposed for 2, 4,

8, 16, and 24 h to different concentrations (125, 250,

500, 1,000 lg ml-1) of the saponin solutions and

mortality rates evaluated after each exposure times.

Phytochem Rev

123972

Ta

ble

1S

apo

nin

sid

enti

fied

inM

.sa

tiva

Agly

cone

C-3

Gly

cosy

lati

on

C-2

8G

lyco

syla

tion

Ref

eren

ces

HO

CO

OH

OH

3

28

Hed

erag

enin

b-D

-Glc

(1?

2)-

a-L

-Ara

–T

imbek

ova

etal

.(1

996)

b-D

-Glc

(1?

2)-

a-L

-Ara

b-D

-Glc

Tim

bek

ova

etal

.(1

993)

b-D

-Gal

(1?

2)-

a-L

-Ara

b-D

-Glc

Mas

siot

etal

.(1

988

)

b-D

-Glc

(1?

3)-

b-D

-Xyl

b-D

-Glc

Tim

bek

ova

etal

.(1

993)

a-L

-Ara

(1?

2)-

b-D

-Glc

(1?

2)-

a-L

-Ara

–T

imbek

ova

and

Abubak

irov

(1985)

a-L

-Ara

(1?

2)-

b-D

-Glc

(1?

2)-

a-L

-Ara

b-D

-Glc

Tim

bek

ova

and

Abubak

irov

(1986a)

HO

CO

OH

HO

OH

Bay

ogen

in

b-D

-Gal

(1?

2)-

b-D

-Glc

Ab

-D-G

lcB

ialy

etal

.(1

999)

HO

OC

HO

CO

OH

HO

Med

icag

enic

aci

d

–b

-D-X

yl(

1?

4)-

a-L

-Rha(

1?

2)-

a-L

-Ara

Mas

siot

etal

.(1

991

)

b-D

-Glc

–M

orr

iset

al.

(1961

)

b-D

-Glc

A–

Ole

szek

etal

.(1

990

)

b-D

-Glc

b-D

-Glc

Tim

bek

ova

and

Abubak

irov

(1984)

b-D

-Glc

a-L

-Rha(

1?

2)-

a-L

-Ara

Tim

bek

ova

etal

.(1

989)

b-D

-Glc

Aa-

L-R

ha(

1?

2)-

a-L

-Ara

Ole

szek

etal

.(1

992a,

b)

b-D

-Glc

b-D

-Xyl(

1?

4)-

a-L

-Rha(

1?

2)-

a-L

-Ara

Tim

bek

ova

and

Abubak

irov

(1986b

)

b-D

-Glc

Ab

-D-X

yl(

1?

4)-

a-L

-Rha(

1?

2)-

a-L

-Ara

Ole

szek

etal

.(1

990

)

b-D

-Glc

AM

ees

ter

b-D

-Xyl(

1?

4)-

a-L

-Rha(

1?

2)-

a-L

-Ara

Bia

lyet

al.

(1999)

a-D

-Glc

(1?

4)-

b-D

-Glc

–L

evy

etal

.(1

989

)

b-D

-Glc

(1?

3)-

b-D

-Glc

b-D

-Glc

Bia

lyet

al.

(1999)

b-D

-Gal

(1?

2)-

b-D

-Glc

b-D

-Glc

Mas

siot

etal

.(1

988

)

b-D

-Glc

(1?

2)-

b-D

-Glc

b-D

-Xyl(

1?

4)-

a-L

-Rha(

1?

2)-

a-L

-Ara

Mas

siot

etal

.(1

988

)

a-L

-Rha(

1?

6)-b

-D-G

lcA

(1?

2)-

b-D

-Glc

–M

orr

isan

dH

uss

ey(1

965)

b-D

-Glc

(1?

6)-

b-D

-Glc

(1?

3)-

b-D

-Glc

–G

este

tner

(1971

)

a-L

-Rha(

1?

2)-b

-D-G

lc(1?

2)-

b-D

-Glc

–B

ialy

etal

.(1

999)

a-L

-Rha(

1?

2)-b

-D-G

lc(1?

2)-

b-D

-Glc

b-D

-Glc

Mas

siot

etal

.(1

988

)

b-D

-Glc

(1?

2)-

b-D

-Glc

(1?

2)-

b-D

-Glc

b-D

-Glc

Bia

lyet

al.

(1999)

b-D

-Glc

(1?

2)-

b-D

-Glc

(1?

2)-

b-D

-Glc

b-D

-Xyl(

1?

4)-

a-L

-Rha(

1?

2)-

a-L

-Ara

Bia

lyet

al.

(1999)

b-D

-Glc

(1?

2)-

b-D

-Glc

(1?

2)-

b-D

-Glc

b-D

-Xyl(

1?

4)-

a-L

-Rha(

1?

2)-

a-L

-Ara

b-D

-Api(

1?

3)

Bia

lyet

al.

(1999)

Phytochem Rev

123973

Ta

ble

1co

nti

nu

ed

Agly

cone

C-3

Gly

cosy

lati

on

C-2

8G

lyco

syla

tion

Ref

eren

ces

b-D

-Glc

(1?

2)-

b-D

-Glc

(1?

2)-

b-D

-Glc

a-L

-Rha(

1?

3)

b-D

-Glc

Tim

bek

ova

etal

.(1

990)

Glc

-mal

onyl

–H

uhm

anan

dS

um

ner

(2002)

Glc

-mal

onyl

Glc

Huhm

anan

dS

um

ner

(2002)

HO

OC

HO

CO

OH

HO

OH

Zha

nic

acid

b-D

-Glc

(1?

2)-

b-D

-Glc

(1?

2)-

b-D

-Glc

b-D

-Xyl(

1?

4)-

a-L

-Rha(

1?

2)-

a-L

-Ara

Bia

lyet

al.

(1999)

b-D

-Glc

(1?

2)-

b-D

-Glc

(1?

2)-

b-D

-Glc

b-D

-Xyl(

1?

4)-

a-L

-Rha(

1?

2)-

a-L

-Ara

b-D

-Api(

1?

3)

Bia

lyet

al.

(1999)

b-D

-Glc

(1?

2)-

b-D

-Glc

(1?

2)-

b-D

-Glc

b-D

-Api(

1?

3)-

b-D

-Xyl(

1?

4)-

a-L

-Rha

23

CO

OH

subst

itute

d:a-

L-A

ra

Ole

szek

etal

.(1

992a,

b)

b-D

-Glc

(1?

2)-

b-D

-Glc

(1?

2)-

b-D

-Glc

b-D

-Api(

1?

3)-

b-D

-Xyl(

1?

4)-

a-L

-Rha

-(1?

2)-

a-L

-Ara

23

CO

OH

subst

itute

d:a-

L-A

ra

Ole

szek

etal

.(1

992a,

b)

HO

OH

OH

OH

Soya

sapo

geno

l A

a-L

-Rha(

1?

2)-b

-D-G

al(1?

2)-

b-D

-Glc

Aa-

L-R

ha

Bia

lyet

al.

(1999)

HO

OH

OH

Soya

sapo

geno

l B

b-D

-Glc

(1?

2)-

b-D

-Glc

A–

Kit

agaw

aet

al.

(1988)

a-L

-Rha(

1?

2)-b

-D-G

lu(1?

2)-

b-D

-Glc

A–

Kit

agaw

aet

al.

(1988)

a-L

-Rha(

1?

2)-b

-D-G

al(1?

2)-

b-D

-Glc

A–

Kit

agaw

aet

al.

(1988)

HO

OH

O

Soya

sapo

geno

l E

a-L

-Rha(

1?

2)-b

-D-G

al(1?

2)-

b-D

-Glc

A–

Kit

agaw

aet

al.

(1988)

a-L

-Rha(

1?

2)-b

-D-G

al(1?

2)-

b-D

-Glc

A22-O

-mal

tol

Mas

siot

etal

.(1

992)

Phytochem Rev

123974

Saponins from both plant parts were found to be

nematotoxic to all the three phytoparasite species and

their activity was dependent on the concentration and

nematode incubation time (Fig. 1; Tables 2, 3).

Exposure for 16 or 24 h at the concentration of

500 lg ml-1 of both saponin mixtures induced

90–100% mortality of X. index and M. incognita,

with no significant difference between top and root

saponins (Fig. 1; Table 2). No significant increase of

nematode mortality could be observed at the highest

saponin concentration (1,000 lg ml-1). At short

exposure times, top saponins were in general the

most active against X. index, inducing a 100%

mortality already after 8 h at 500 lg ml-1.

Root saponins were not nematotoxic to M. incog-

nita at the lower concentrations, whereas they caused

48% mortality of X. index at 250 lg ml-1 (Fig. 1;

Table 2).

Very interestingly, parallel experiments on

X. index with saponins from M. sativa and a

commercial saponin (90%) extract from Q. saponaria

bark, showed that saponins from alfalfa had a

significantly higher nematotoxic effect, as quillay

saponins resulted in a maximum mortality rate of

only 19–24% (Argentieri et al. 2008).

Second-stage juveniles of G. rostochiensis resulted

the most susceptible to the biocidal effect of M. sativa

saponins, as 40–54% mortality rates were found

between 4 and 8 h at 125 lg ml-1. However, maxi-

mum mortality rates ranged between 68–91% and

57–89% after 24 h exposure at 250–1,000 lg ml-1 of

top and root saponins, respectively (Table 3).

Biological effects of saponins are normally

ascribed to their specific interaction with the cell

membranes causing changes in the cell permeability

(Hostettmann and Marston 1995; Oleszek 1996;

Sprag et al. 2004; Tava and Avato 2006).

A critical structure for nematode viability is the

protective cuticle that forms the organism exoskele-

ton and primarily consists of collagen proteins

assembled into higher order complexes (Spiegel and

McClure 1995; Page and Winter 2003) which can

possibly be involved in the mechanism of activity.

Bird and Bird (1991) reported differences in the

chemical composition and ultrastructure of the cuticle

among the different nematode species, which may

result in different interaction with saponin molecules

and, therefore, give account for different susceptibil-

ity of tested nematode species to M. sativa saponins.

Structure–activity relationship

Saponins effect on the cell membranes was reported

to be affected by side sugar chains attached to the

sapogenin moiety, as monodesmoside saponins have

been generally shown to be more bioactive than the

related bidesmosides (Oleszek et al. 1990; Tava and

Avato 2006).

Nevertheless, a specific study (Avato et al. 2006)

on the antimicrobiotic potential of saponins from

0

20

40

60

80

100

Mor

talit

y %

2 4 8 16 24 2 4 8 16 24 2 4 8 16 24

500250125

µg ml-1

Tops Roots

Fig. 1 Effect of tested concentrations of saponins from

M. sativa tops and roots on X. index after different exposure

times

Table 2 Effect of saponins from top and root tissues of

M. sativa on the mortality of second-stage juveniles

M. incognita after different exposure times

Treatment Dose

(lg ml-1)

Mortality %

2 h 4 h 8 h 16 h 24 h

Saponin tops 125 1.3ab 0.6ab 3.6c 3.4b 3.6b

Saponin tops 250 2.2b 1.0abc 2.1bc 4.6b 6.8b

Saponin tops 500 0.3a 1.8abc 2.5bc 91.7c 94.0c

Saponin tops 1,000 2.2b 1.3bc 2.8bc 93.4c 99.0d

Saponin roots 125 0.6ab 3.3c 1.8bc 4.4b 6.5b

Saponin roots 250 0.5a 1.8bc 0.4ab 1.9b 3.1b

Saponin roots 500 0.7ab 2.0bc 1.0abc 88.3c 98.0cd

Saponin roots 1,000 1.6ab 2.6bc 1.4abc 86.9c 98.2d

Water – 0.3a 0.1a 0.0a 0.0a 0.3a

Data are means of four replicates

Means followed by the same letters on the same column are not

significantly different (P B 0.05) according to Least Significant

Difference Test

Phytochem Rev

123975

Medicago showed that the presence of sugars might

not be a requisite for the activity against human

pathogens since it was found that the antibiotic effect

increased from the raw saponin mixture to the

prosapogenin and the related sapogenins.

A comparative study of the in vitro activity of

M. sativa top saponins (mainly bidesmosidic), root

saponins (mostly monodesmosidic), and related pros-

apogenins (after basic hydrolysis) and sapogenins

(after acid hydrolysis) was carried out on X. index

(Argentieri et al. 2008), M. incognita and

G. rostochiensis.

Data reported by Argentieri et al. (2008) showed

that prosapogenins from M. sativa aerial parts were

more nematicidal against X. index than the related

saponins at the same dose, except for the maximal

concentration (Fig. 2). Toxicity of tops saponins was

in fact consistent only at 500 lg ml-1, while pros-

apogenins were already nematotoxic at 125 lg ml-1

and, at this dose, their activity almost overlapped that

of the related sapogenins, progressively increasing at

the two higher concentrations. Compared to sapoge-

nins, the nematicidal efficacy of prosapogenins from

tops was evident after a shorter time of treatment, as

8 h treatment at the dose of 250 lg ml-1 resulted in a

57% mortality.

Medicagenic acid, the dominant aglycone in

M. sativa saponins, was also assayed as pure compound;

it displayed a good activity, with a trend rate comparable

with that of top sapogenin mixtures (Fig. 2).

No straight structure–activity relationship was

evidenced in the experiments with M. incognita

(Table 4). Sapogenins from both tops and roots were

already active at 125 and 250 lg ml-1, though

mortality rates did not exceed 20% also after a 24 h

treatment, whereas a 16 h exposure to 500 and

1,000 lg ml-1 solutions of top and root saponins

resulted in almost 90% juvenile mortality. A very low

nematotoxic activity on M. incognita was always

detected for top prosapogenins and medicagenic acid

(Table 4).

Saponins from M. sativa resulted particularly toxic

to G. rostochiensis and a very high nematicidal

activity was observed also for top prosapogenins

(Table 5). Mortality rates of G. rostochiensis juve-

niles were 34–52% just after a 2 h exposure to

prosapogenin solutions and progressively increased

by rate and exposure time up to 60–65% (Table 5).

Very interestingly, prosapogenin activity was on

average more similar to that of root than top saponins,

as expected based on their structural similarity.

Mortality of G. rostochiensis was consistent also in

the sapogenin solutions, though never higher than in

saponin and prosapogenin solutions at corresponding

concentrations and exposure times. Root sapogenins

were more nematotoxic than top sapogenins, as

causing 23–31% nematode mortality already after

2 h of incubation at 250–1,000 lg ml-1.

Medicagenic acid displayed activity rates in good

correlation with that of prosapogenins and root

Table 3 Effect of saponins from top and root tissues of M. sativa on the mortality of second-stage juveniles of G. rostochiensis after

different exposure times

Treatment Dose (lg ml-1) Mortality %

2 h 4 h 8 h 16 h 24 h

Saponin tops 125 13.1b 53.4c 54.1c 59.4cd 65.8d

Saponin tops 250 39.8 cd 57.4cd 59.5d 63.3d 68.5d

Saponin tops 500 47.7de 68.0e 64.1e 74.6f 77.2e

Saponin tops 1,000 58.3f 63.8de 70.6g 73.8f 90.7f

Saponin roots 125 31.2c 40.1b 44.3b 45.4b 47.4b

Saponin roots 250 39.4cd 51.8c 53.9c 55.3c 57.4c

Saponin roots 500 49.6ef 58.1cd 63.2de 64.5de 66.3d

Saponin roots 1,000 55.1ef 60.4cde 65.8ef 69.5ef 89.0f

Water – 0.3a 0.1a 0.0a 0.0a 0.3a

Data are means of four replicates

Means followed by the same letters on the same column are not significantly different (P B 0.05) according to Least Significant

Difference Test

Phytochem Rev

123976

saponins, determining 32–62% mortalities of

G. rostochiensis juveniles through all the rates and

exposure times (Table 5). More generally, G. rosto-

chiensis resulted highly susceptible to medicagenic

acid, as similar rates of toxicity were not observed on

the other two nematode species used in this

investigation.

Data obtained in this study on the structure–

activity relationship between saponins and related

prosapogenins and sapogenins cannot be straight

razionalized and suggest that further investigations

are needed. However, data from the experiments on

X. index and G. rostochiensis seem to indicate that

M. sativa prosapogenins (monodesmosides) can be

more nematotoxic than the related saponins (bides-

mosides), and more or as much as active than the

related sapogenins, though physiological differences

among the target nematode species may also con-

tribute to the overall efficacy of the drug. Assays with

medicagenic acid, the main aglycone present in the

saponin mixtures from M. sativa also suggest that this

molecule is highly implicated in the detected nem-

aticidal bioactivity.

Efficacy of soil amendments

Biological and chemical mechanisms, either alone

or in combination, have been reported to play a

role in nematode suppression by organic amend-

ments (Akhtar and Malik 2000). Allelochemicals

are often produced in large amounts in plant

material or in agriculture wastes and the use of

organic amendments is an effective means for the

release of such compounds in the soil (Kokalis-

Burelle and Rodriguez-Kabana 2006). Moreover, a

direct relationship between nitrogen content of

organic amendments and their nematicidal activity,

due to the release of toxic ammoniacal compounds

is also reported in the literature (Bailey and

Lazarovitis 2003).

Bioactivity data obtained in vitro experiments with

M. sativa saponin mixtures (Avato et al. 2006;

Argentieri et al. 2008) and the high nitrogen content

of this crop suggested us to explore the efficacy of

Medicago material to suppress plant parasitic nema-

todes through soil amendments (D’Addabbo et al.

2009).

Trials were conducted either on tomato and potato

in potting mixes infested with M. incognita or

G. rostochiensis, respectively, and amended with

different rates (0.5, 1.0, 2.0, 4.0 g kg-1 soil) of dry

top or root material from M. sativa, previously

characterized for the total content of saponins,

phenolics, nitrogen and carbon (Table 6). Suppres-

sivity of different amendment rates was evaluated on

the base of root and soil nematode population and

gall formation on tomato roots for M. incognita, and

of the cyst and egg soil density for G. rostochiensis

(D’Addabbo et al. 2009).

All doses of either leaf and root amendments were

found to reduce root and soil population densities of

M. incognita compared to a non-treated control, and

125 µg ml-

0

20

40

60

80

100

120

4824842

Exposure time (h)

250 µg ml-1

0

20

40

60

80

100

120

Exposure time (h)

500 µg ml-

0

20

40

60

80

100

120

Exposure time (h)

Saponins Tops Saponins Roots Prosapogenins TopsSapogenins Tops Sapogenins Roots MedA

4824842

4824842

Fig. 2 Nematicidal activity of saponins, prosapogenins, sapo-

genins from M. sativa tops and roots and medicagenic acid on

X. index

Phytochem Rev

123977

the effect was found to increase with the amendment

concentration (Fig. 3). In addition, the incorporation

of M sativa plant materials resulted also in a larger

growth of tomato plant top and roots compared either

to non-treated and fenamiphos-treated soil (Fig. 3;

D’Addabbo et al. 2009). No significant differences in

suppressivity were observed between treatments with

top or root plant material.

Table 4 Effect of

prosapogenins and

sapogenins from top and

root tissue of M. sativa and

medicagenic acid on the

mortality of second-stage

juveniles of M. incognitaafter different exposure

times

Data are means of four

replicates

Means followed by the

same letters on the same

column within each product

(prosapogenin, sapogenins

and medicagenic acid) are

not significantly different

(P B 0.05) according to

Least Significant Difference

Test

Treatment Dose (lg ml-1) Mortality %

2 h 4 h 8 h 16 h 24 h

Prosapogenin tops 125 2.6b 3.1b 3.8b 4.5b 4.8b

Prosapogenin tops 250 2.7b 2.9b 3.9b 4.6bc 5.3b

Prosapogenin tops 500 2.5b 2.8b 4.8bc 5.3cd 5.8bc

Prosapogenin tops 1,000 2.8b 3.3b 5.3c 5.8d 6.8c

Sapogenin tops 125 1.0ab 2.7b 2.6b 2.8ab 10.66b

Sapogenin tops 250 1.9abc 3.0b 4.7c 5.6bc 20.4cd

Sapogenin tops 500 2.3bc 4.8cd 9.4d 11.6e 21.8de

Sapogenin tops 1,000 3.1cd 6.1de 13.8e 17.4f 23.6ef

Sapogenin roots 125 1.6abc 2.7b 4.7c 6.4c 18.1c

Sapogenin roots 250 2.6c 3.2bc 5.4c 7.9cd 19.4cd

Sapogenin roots 500 2.9cd 4.7cd 7.8d 9.7de 20.8d

Sapogenin roots 1,000 4.4d 6.9e 12.2e 16.6f 26.6g

Medicagenic acid 125 0.6a 1.2b 1.3b 2.5b 3.6b

Medicagenic acid 250 0.7a 1.5b 2.2c 3.1b 4.1b

Medicagenic acid 500 1.6b 3.3c 3.9d 5.6c 6.4c

Medicagenic acid 1,000 2.2b 4.5d 4.7d 5.9c 7.3c

Water – 0.2a 0.0a 0.0a 0.2a 0.3a

Table 5 Effect of

prosapogenins and

sapogenins from top and

root tissue of M. sativa and

medicagenic acid on the

mortality of second-stage

juveniles of G.rostochiensis after different

exposure times

Data are means of four

replicates

Means followed by the

same letters on the same

column within each product

(prosapogenin, sapogenins

and medicagenic acid) are

not significantly different

(P B 0.05) according to

Least Significant Difference

Test

Treatment Dose (lg ml-1) Mortality %

2 h 4 h 8 h 16 h 24 h

Prosapogenin tops 125 33.9b 47.9b 52.8b 55.4b 57.5b

Prosapogenin tops 250 37.2b 52.1b 55.5bc 59.3c 60.4bc

Prosapogenin tops 500 49.0c 54.8b 58.8cd 60.2cd 62.4cd

Prosapogenin tops 1,000 52.2c 57.1b 61.5d 62.3d 64.8d

Sapogenin tops 125 2.0a 5.8b 14.5b 22.1b 28.3b

Sapogenin tops 250 7.5b 10.1c 22.3c 28.4c 38.6c

Sapogenin tops 500 12.1c 23.7e 27.3d 33.3d 40.2cd

Sapogenin tops 1,000 16.7d 30.7g 33.2e 39.2e 48.2ef

Sapogenin roots 125 12.5c 15.8d 37.0ef 40.1e 41.9d

Sapogenin roots 250 23.1e 26.8f 39.7fg 42.7f 47.0e

Sapogenin roots 500 26.7f 31.9gh 41.4fg 44.4fg 49.4fg

Sapogenin roots 1,000 30.8g 35.0i 44.0g 48.0h 53.9h

Medicagenic acid 125 31.5b 33.5b 36.4b 39.2b 42.3b

Medicagenic acid 250 35.6bc 39.6c 40.4b 42.2c 44.8b

Medicagenic acid 500 39.3cd 43.2cd 46.6c 50.0d 57.7c

Medicagenic acid 1,000 42.5d 47.2d 52.3d 57.0e 61.6c

Water 1.6a 3.1a 1.3a 1.1a 1.1a

Phytochem Rev

123978

Moreover, the root system from tomato plants

grown in alfalfa-amended soil showed a normal

development and a significantly lower number of

galls due to M. incognita infestation compared to the

control plants (Fig. 4).

Similar results were obtained on G. rostochiensis,

as number of nematode cysts and eggs were always

significantly lower in the soil amended with M. sativa

top meal than in the control with significant differ-

ences among the amendments rates (D’Addabbo et al.

2009). A significant analytical relationship between

nematode mortality and amendment rate was esti-

mated for both nematode species (Fig. 5).

Further field experiments evidenciated the high

suppressiveness of a pelleted formulation of M. sativa

dry biomass on M. incognita on tomato and on the

cyst nematode H. carotae on carrot. (Table 7;

D’Addabbo et al. 2009a, b). Soil population density

of both nematodes was significantly lower in soil

amended with 20 and 40 t ha-1 alfalfa pelleted meal

than in soil non-treated or treated with a commercial

quillay formulation (Tables 7, 8). Moreover, sup-

pressive effect of the highest amendment rate was not

statistically different from that of the chemical

nematicide fenamiphos.

Very few data on the nematicidal activity of soil

amendments with M. sativa plant material were

Table 6 Total content of saponins, phenolics, nitrogen and

carbon of M. sativa plant samples

Plant

material

Total

saponins

(% dw)

Total phenolics*

(mg g-1dw)

Total N

(% dw)

Total C

(% dw)

Tops 2.1 ± 0.3 8.5 ± 0.3 29.2 42.5

Roots 1.9 ± 0.2 2.9 ± 0.1 12.7 43.2

Pellet 0.8 ± 0.3 5.1 ± 0.5 19.5 40.4

* Gallic acid equivalent

Each value was obtained by calculating the average of three

determinations ± standard deviation

Fig. 3 Effect of M. sativa soil amendments on the population of M. incognita on tomato roots (a), in the soil (b), on gall formation

(c) and on tomato root weight (d)

Phytochem Rev

123979

available before our experiments, as only Walker

(2007) reported the suppressive effect of soil amend-

ment with 15 t ha-1 alfalfa hay pellet on M. javanica

in pot experiments.

However, results suggest that saponin content of

M. sativa tissues can be considered only partially

responsible for phytonematode suppression in

amended soil and the contributory effect of other

different mechanisms should be hypothesized. Active

metabolites other than saponins, such as phenolics

and canavanine (Natelson 1985), are present in

M. sativa tissues, and may contribute to the nema-

ticidal effect. Moreover, the low C/N ratio of

M. sativa materials may suggest also the contribution

of ammoniacal nitrogen to nematode suppression in

soil, as the release of ammonia during decomposition

of low C/N ratio amendments, such as legume crop

green manures, was found to be suppressive on soil

phytoparasitic nematophauna (Bailey and Lazarovits

2003; Jaffee 2006; Jansen and McGinn 1991).

Finally, the presence of a biological mechanism

should be also hypothesized, as soil population of

phytonematode-suppressive microorganisms was

found to increase following soil incorporation of

organic amendments, among which also alfafa bio-

mass (Jaffee 2006).

Incorporation of M. sativa pellets in field soil

resulted also in a significant increase of tomato and

Fig. 4 Tomato roots from soil uninfested (a), treated with 20 t ha-1 M. sativa dry leaves (b), treated with fenamiphos (c) and

untreated (d)

Phytochem Rev

123980

carrot yield compared to non-treated, and fenamiphos

or quillay formulation (Tables 7, 8). Improvement of

plant growth and yield performances following the

alfalfa amendments can be only partially related to

phytonematode suppression by saponins and possibly

other active metabolites, but it should be also related

to the improved physical, chemical and microbiolog-

ical soil properties following the incorporation of

organic amendments (Bulluck et al. 2002) as well as

to the intake of large nitrogen amounts.

Conclusions

Data obtained with M. sativa saponins support their

potential use for new biotechnological applications

such as nematicidal formulations, though biological

activity of these saponins is related to their chemical

structures and may change according to the target

nematode species.

Large biomass produced by Medicago species

could make the industrial extraction of saponins

economically viable, as demonstrated by the com-

mercial exploitation of Q. saponaria extracts formu-

lations already commercially available.

Soil amendments with M. sativa pelleted meal

demonstrated to be strongly suppressive either on

root-knot and cyst nematode species also in the

presence of heavy infestation conditions and, there-

fore, it could be hypothesized as a valuable option for

an environmentally safe nematode management. A

critical point for a farm-level application of this

technique may be represented by its cost at the tested

dosages, though cost-benefit analysis should consider

positive effects on plant growth and crop yield and

the environmental benefits related to the withdrawal

of chemical treatments. However, the available

results (Walker 2007) showed that alfalfa amend-

ments may work also at lower concentrations, as well

as a reduction of amendment rates may be also

Fig. 5 Relationship between rates (x) of amendments with

M. sativa plant material and percent mortality (y) of

M. incognita (filled circle) and G. rostochiensis (filled square)

Table 7 Effect of soil amendments with M. sativa pellets in field on infestation parameters of M. incognita on tomato and H. carotaeon carrot

Treatment Dose M. incognita on tomato H. carotae on carrot

Eggs and juveniles

(ml-1 soil)

Reproduction

rate

Root gall

index

Eggs and juveniles

(g-1 soil)

Reproduction

rate

M. sativa pellet 20 t ha-1 3.2c 2.3b 1.9c 11.6bc 0.8bc

M. sativa pellet 40 t ha-1 1.5d 0.8d 1.3d 7.5c 0.5c

Q. saponaria extract 30 l ha-1 3.8bc 3.0a 3.1b 22.3ab 1.6ab

Fenamiphos 30 l ha-1 2.7cd 1.6c 1.8cd 8.6c 0.6c

Control – 5.2a 3.3a 4.0a 32.0a 2.2a

Data are means of five replicates

Means followed by the same letters on the same column are not significantly different (P B 0.05) according to Least Significant

Difference Test

Phytochem Rev

123981

achieved through the combination with other sup-

pressive materials (i.e. biofumigant brassicas or farm

manures) or other nonchemical techniques, as soil

solarization.

Finally, exploitation of potential M. sativa saponin

formulations, as well as of alfalfa meal soil amend-

ments, seems to be particularly suitable to organic

agriculture but can be easily extended also to

conventional farms.

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Treatment Dose Trial on M. incognita Trial on H. carotae

Tomato yield (t ha-1) Total carrot yield (t ha-1) Marketable %

M. sativa pellet 20 t ha-1 68.6c 75.6b 50.3b

M. sativa pellet 40 t ha-1 71.6c 76.0b 68.5c

Q. saponaria extract 30 l ha-1 55.5b 59.6a 43.0b

Fenamiphos 30 l ha-1 58.3b 107.2c 63.9c

Control – 46.0a 58.2a 29.7a

Data are means of five replicates

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