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: [email protected]
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
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1?
2)-
a-L
-Ara
Mas
siot
etal
.(1
991
)
b-D
-Glc
–M
orr
iset
al.
(1961
)
b-D
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A–
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szek
etal
.(1
990
)
b-D
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bek
ova
and
Abubak
irov
(1984)
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a-L
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1?
2)-
a-L
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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(
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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
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Kit
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-D-G
al(1?
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-Glc
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agaw
aet
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Soya
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geno
l E
a-L
-Rha(
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2)-b
-D-G
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agaw
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-D-G
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-Glc
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-mal
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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.
References
Akhtar M (2000) Nematicidal potential of the neem tree
Azadirachta indica (A. Juss). Integr Pest Manage Rev
5:57–66
Akhtar M, Malik A (2000) Roles of organic soil amendments
and soil organisms in the biological control of plant-par-
asitic nematodes: a review. Biores Technol 74:35–47
Argentieri MP, D’Addabbo T, Tava A, Agostinelli A, Jurzysta
M, Avato P (2008) Evaluation of nematicidal properties of
saponins from Medicago ssp. EJPP 120:189–197
Avato P, Bucci R, Tava A, Vitali C, Rosato A, Bialy Z, Jur-
zysta M (2006) Antimicrobial activity of saponins from
Medicago sp.: structure–activity relationship. Phytoth Res
20:454–457
Avato P, Migoni D, Fanizzi FP, Tava A (2008) Cytotoxic
effect of saponins from Medicago on tumor cellular lines.
In: International Symposium PSE—Natural Products in
Cancer Therapy, Abstract Book, p 11
Bader G, Plohmann B, Hiller K, Franz F (1996) Cytotoxicity of
triterpenoid saponins. Part 1: activities against tumor cells
in vitro and haemolytic index. Pharmazie 51:414–417
Bailey KL, Lazarovitis G (2003) Suppressing soil-borne dis-
eases with residue managment and organic amendments.
Soil Tillage Res 72:169–180
Bar-Eyal M, Sharon E, Spiegel Y (2006) Nematicidal activity
of Chrysanthemum coronarium. EJPP 114:427–433
Barnes J, Anderson LA, Phillipson JD (2002) Herbal medi-
cines. Pharmaceutical Press, London
Batish DR, Singh HP, Kohli RK, Kaur S (2008) Eucalyptus
essential oil as a natural pesticide. Forest Ecol Manage
256:2166–2174
Behrens E (1975) Globodera Skarbilovich, 1959 an indepen-
dent genus in the subfamily Heteroderinae Skarbilovich,
1949 (Nematoda: Heteroderidae). Vortragstagung zu
Aktuellen Problemen der Phytonematologie 1:12–26
Berrang B, Davis KH Jr, Wall ME, Hanson CH, Pedersen ME
(1974) Saponins of two alfalfa cultivars. Phytochemistry
13:2253–2260
Bialy Z, Jurzysta M, Oleszek W, Piacente S, Pizza C (1999)
Saponins in alfalfa (Medicago sativa L.) root and their
structural elucidation. J Agric Food Chem 47:3185–3192
Bialy Z, Jurzysta M, Mella M, Tava A (2004) Triterpene
saponins from aerial parts of Medicago arabica L. J Agric
Food Chem 52:1095–1099
Bialy Z, Jurzysta M, Mella M, Tava A (2006) Triterpene
Saponins from the Roots of Medicago hybrida. J Agric
Food Chem 54:2520–2526
Bilgrami AL (1997) Nematode biopescticides. Aligarh Uni-
versity Press, Aligarh
Bird AF, Bird J (1991) The structure of nematodes, 2nd edn.
Academic Press, San Diego
Brown EB (1969) Assessment of the damage caused to pota-
toes by potato cyst eelworm Heterodera rostochiensisWoll. Ann Appl Biol 63:493–502
Brown DJF, Robertson WM, Trudgill DL (1995) Transmission
of viruses by plant nematodes. Ann Rev Phytopathol
33:223–249
Bruneton J (1999) Pharmacognosy—phytochemistry, medici-
nal plants. Lavoisier Publishing, Paris, pp 671–719
Bulluck LR, Brosius M, Evanylo GK, Ristaino JB (2002)
Organic and synthetic fertility amendments influence soil
microbial, physical and chemical properties on organic
and conventional farms. Appl Soil Ecol 19:147–160
Cheeke PR (1996) Biological effects of feed and forage sap-
onins and their impacts on animal production. In: Waller
GR, Yamasaky K (eds) Advances in experimental medi-
cine and biology. Saponins used in food and agriculture,
vol 405. Plenum Press, New York, pp 377–385
Table 8 Effect of soil amendments with M. sativa pellets in field on tomato and carrot yield in soil infested by M. incognita and H.carotae, respectively
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
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
123982
Chitwood DJ (2002) Phytochemical based strategies for nem-
atode control. Ann Rev Phytopathol 40:221–249
D’Addabbo T, Curto G, Greco P, Di Silvestro D, Coiro MI,
Lamberti F, Ferrari V, Santi R, Carella A (2005) Prove
preliminari di Lotta contro nematodi galligeni mediante
estratti di Quillaja saponaria Molina. Nem Med 33:29–34
D’Addabbo T, Avato P., Sasanelli N, Agostinelli A, Radicci V
(2007) Biocidal activity of glucosinolates on phytopara-
sitic nematodes. In: Tenth international helminthological
symposium—helminths, helminthoses and environment,
p. 66
D’Addabbo T, Avato P, Radicci V, Tava A (2009) Soil
amendments with pelleted plant material of Medicagosativa for the controlof root-knot and cyst nematodes. In:
VII International symposium on chemical and non-
chemical soil and substrate disinfestation, 13–17 Sep-
tember 2009, Leuven, Belgium. Abstract Book, p 82
D’Addabbo T, Avato P, Tava A (2009b) Nematicidal potential
of materials from Medicago spp. Eur J Plant Pathol
125:39–49
Duke SO (1990) Natural pesticides from plants. http://www.
hort.purdue.edu/newcrop/proceedings1990/v1-511html
Evron R, Polacheck I, Guizie M, Levy M, Zehavi U (1988)
Activities of compound G2 isolated from alfalfa roots
against dermatophytes. Antimicrob Agents Chemother
32:1586–1587
Gestetner B (1971) Structure of a saponin from lucerne
(Medicago sativa). Phytochemistry 10:2221–2223
Gestetner B, Assa Y, Henis Y, Birk Y, Bondi A (1971) Lucerne
saponins IV: relationship between their chemical consti-
tution, and haemolytic and antifungal activities. J Agric
Food Chem 22:168–172
Ghorbani R, Wilcockson S, Koochek A, Leifert C (2008) Soil
management for sustainable crop disease control: a
review. Environ Chem Lett 6:149–162
Gonzalez JA, Estevez-Braun A (1997) Phytonematicidal
activity of aromatic compounds related to shikimate
pathway. Pest Biochem Physiol 58:193–197
Greco N, D’Addabbo T, Brandonisio A, Elia F (1993) Damage
to italian crops caused by cyst-forming nematodes. J
Nematol 25(4S):836–842
Guenzi WD, Kehr WR, McCalla TM (1964) Water-soluble
phytotoxic substances in alfalfa forage:variation with
variety, cutting, year, and stage of growth. Agronomy J
56:499–500
Heyn CC (1963) The annual species of Medicago. Magnes
Press, Hebrew University, Jerusalem
Hoagland RE, Zablotowicz RM, Oleszek WA (2001) Effects of
alfalfa saponins on in vitro physiological activity of soil
rhizosphere bacteria. J Crop Prod 4:349–361
Hostettmann K, Marston A (1995) Chemistry and pharmacol-
ogy of natural products. In: Phillipson JD, Baxter H (eds)
Saponins. Cambridge University Press, UK
Houghton P, Patel N, Jurzysta M, Bialy Z, Cheung C (2006)
Antidermatophyte activity of medicago extraccts con-
tained saponins and their structure–activity relationships.
Phytother Res 20:1061–1066
Huang HC, Wu MD, Tsai WJ, Liao SC, Liaw CC, Hsu LC, Wu
YC, Kuo YH (2008) Triterpenoid saponins from the fruits
and galls of Sapindus mukorossi. Phytochemistry 69:
1609–1616
Huhman DV, Sumner LW (2002) Metabolic profiling of sap-
onins in Medicago sativa and Medicago truncatula using
HPLC coupled to an electrospray ion-trap mass spec-
trometer. Phytochemistry 59:347–360
Huhman DV, Berhow MA, Sumner LW (2005) Quantification
of saponins in aerial and subterranean tissues of Medicagotruncatula. J Agric Food Chem 53:1914–1920
Hynes RK, Boyetchko SM (2006) Research initiatives in the
art and science of biopesticide formulations. Soil Biol
Biochem 38:845–849
Jaffee BA (2006) Interactions among soil organic amendments,
nematodes and the nematode-trapping fungus Dactylellinacandidum. Phytopathology 96:1388–1396
Jansen HH, McGinn S (1991) Volatile loss of nitrogen during
decomposition of legume green manure. Soil Biol Bio-
chem 23:291–297
Javed N, Gowen SR, El-Hassan SA, Inam-ul-Haq M, Shahina
F, Pembroke B (2008) Efficay of neem (Azadirachtaindica) formulations on biology of root-knot nematodes
(Meloidogyne javanica) on tomato. Crop Prot 27:36–43
Jurzysta M, Bialy Z (1999) Antifungal and haemolytic activity
of roots of alfalfa (Medicago spp.) in relation to saponin
composition. In: Lyr H, Russel PE, Sisler HD (eds)
Modern fungicides and antifungal compounds II. Inter-
cept, Andover, pp 445–451
Jurzysta M, Waller GR (1996) Antifungal and haemolytic
activity of aerial parts of alfalfa (Medicago) species in
relation to saponin composition. In: Waller GR, Yamasaki
K (eds) Advances in experimental medicine and biology.
Saponis used in traditional and modern medicine, vol 404.
Plenum Press, New York, pp 565–574
Kapusta I, Bogdan J, Stochmal A, Oleszek W (2005a) Deter-
mination of saponins in aerial parts of barrel medic
(Medicago truncatula) by liquid chromatography-elec-
trospray ionization/mass spectrometry. J Agric Food
Chem 53:7654–7660
Kapusta I, Stochmal A, Perrone A, Piacente S, Pizza C,
Oleszek W (2005b) Triterpene saponins from barrel medic
(Medicago truncatula) aerial parts. J Agric Food Chem
53:2164–2170
Kitagawa I, Taniyama T, Murakami T, Yoshihara M, Yos-
hikawa M (1988) Saponin and sapogenol. XLVI. On the
constituents of aerial part of american alfalfa, Medicagosativa L. The structure of dehydrosoyasaponin I. Yak-
ugaku Zasshi 108:547–551
Kokalis-Burelle N, Rodriguez-Kabana R (2006) Allelochemi-
cals as biopesticides for managment of plant-parasitic
nematodes. In: Inderjit KG, Mukerji (eds) Allelochemi-
cals: biological control of plant pathogens and diseases.
Springer, The Netherlands, pp 15–29
Kong JO, Lee SM, Moon YS, Lee SG, Ahn YJ (2006) Nem-
aticidal activity of plant essential oils against Bursaphe-lenchus xylophilus (Nematoda: Aphelenchoididae). J
Asia-Pacific Entomol 9:173–178
Kuljanabhagavad T, Wink M (2009) Biological activities and
chemistry of saponins from Chenopodium quinoa Willd.
Phytochem Rev 8:473–490
Lacaille-Dubois MA, Wagner H (2000) Bioactive saponins
from plants: an update. In: Atta-ur-Rahman (ed) Studies in
natural products chemistry, vol 21. Elsevier, London,
pp 633–687
Phytochem Rev
123983
Lamberti F, Taylor CE (eds) (1979) Root-knot nematodes
(Meloidogyne species): systematics, biology and control.
Academic Press, New York
Lesins KA, Lesins I (1979) Genus Medicago (Leguminosae).
In: Junk W (ed) A taxogenetic study. Publisher, London
Levy M, Zehavi U, Naim M, Polacheck I (1989) Isolation,
structure determination and antifungal activity of a new
alfalfa root saponin. Carbohydr Res 193:115–123
Macias FA, Galindo JLG, Garcia-Diaz MD, Galindo JCG (2008)
Allelophatic agents from aquatic ecosystems: potential
biopesticides models. Phytochem Rev 7:155–178
Martyniuk S, Jurzysta M, Bialy Z, Wroblewska B (1995a)
Alfalfa root saponins affect the growth and sporulation of
Cephalosporium gramineum. In: Manka M (ed) Environ-
mental biotic factors in integrated plant disease control.
The Polish Phytopathological Society, Poznan, pp 395–398
Martyniuk S, Wroblewska B, Jurzysta M, Bialy Z (1995b)
Saponins as inhibitors of cereal pathogens: Gaeumanno-myces graminis v. tritici and Cephalosporium gramineum.
In: Lyr H, Russel PE, Sisler HD (eds) Modern fungicides
and antifungal compounds. Intercept Ltd, UK, pp 193–197
Massiot G, Lavaud C, Le Men-Olivier L, van Binst G, Miller
SPF, Fales HM (1988) Structural elucidation of alfalfa
root saponins by mass spectrometry and nuclear magnetic
resonance analysis. J Chem Soc Perkin Trans I:3071–3079
Massiot G, Lavaud C, Besson V, Le Men-Olivier L, van Binst
G (1991) Saponins from aerial parts of alfalfa (Medicagosativa). J Agric Food Chem 39:78–82
Massiot G, Lavaud C, Benkhaled M, Le Men-Olivier L (1992)
Soyasaponin VI, a new maltol conjugate from alfalfa and
soyabean. J Nat Prod 55:1339–1341
Messina MJ, Persky V, Setchell KDR, Barnes S (1994) Soy
intake and cancer risk: a review of the in vitro and in vivo
data. Nutr Cancer 21:113–131
Mishustin BN, Naumova AN (1955) Secretion of toxic sub-
stances by alfalfa and their effect on cotton and soil
microflora. Akademia Nauk USSR Izvestija Ser Biol 6:3–9
Morris RJ, Hussey EW (1965) A natural glycoside of medi-
cagenic acid. An alfalfa blossom saponin. J Org Chem
30:166–168
Morris RJ, Dye WB, Gisler DS (1961) Isolation, purification
and structural activity of an alfalfa root saponins. J Org
Chem 26:1241–1243
Oleszek W (1996) Alfalfa saponins: structure, biological
activity and chemotaxonomy. In: Waller GR, Yamasaki K
(eds) Advances in experimental medicine and biology.
Saponis used in food and agriculture, vol 405. Plenum
Press, New York, pp 155–170
Oleszek W (1998) Composition and quantitation of saponins in
alfalfa (Medicago sativa L.) seedlings. J Agric Chem
46:960–962
Oleszek W (2000) Saponins. In: Naidu AS (ed) Natural food
antimicrobial systems. CRC Press, London, pp 1–30
Oleszek W, Price KR, Fenwick GR (1988) Triterpene saponins
from the roots of Medicago lupulina L. (black medic
trefoil). J Sci Food Agric 43:289–297
Oleszek W, Price KR, Colquhoun IJ, Jurzysta M, Ploszynski
M, Fenwick GR (1990) Isolation and identification of
alfalfa (Medicago sativa L.) root saponins: their activity in
relation to a fungal bioassay. J Agric Food Chem
38:1810–1817
Oleszek W, Jurzysta M, Gorski PM (1992a) Alfalfa saponins-
the allelopathic agents. In: Rizvi SJH, Rizvi V (eds)
Allelopathy: basic and applied aspects. Chapman & Hall,
London, pp 151–167
Oleszek W, Jurzysta M, Ploszynski M, Coloquhoun IJ, Price
KR, Fenwick GR (1992b) Zahnic acid tridesmoside and
other dominant saponins from alfalfa (Medicago sativaL.) aerial parts. J Agric Food Chem 40:191–196
Page AP, Winter AD (2003) Enzymes involved in the bio-
genenesis of the nematode cuticle. Adv Parasitol 53:
85–148
Pecetti L, Tava A, Romani M, De Benedetto MG, Corsi P
(2006) Variety and environment effects on the dynamics
of saponins in lucerne (Medicago sativa L.). Eur J Agron
25:187–192
Pedersen MW, Wang (1971) Modification of saponin content
of alfalfa through selection. Crop Sci 11:833–835
Pedersen MW, Zimmer DE, McAllister DR, Anderson JO,
Wilding MD, Taylor GA, McGuire CF (1967) Compara-
tive studies of several alfalfa varieties using chemical and
biochemical assay. Crop Sci 7:349–352
Pelah D, Abramovich Z, Markus A, Wiesman Z (2002) The use
of commercial saponin from Quillaja saponaria bark as a
natural larvicidal agent against Aedes aegypti and Culexpipiens. J Ethnopha 81:407–409
Polacheck I, Zehavi U, Naim M, Levy M, Evron R (1986)
Activity of compound G2 isolated from alfalfa roots
against medically important yeasts. Antimicrob Agents
Chemother 30:290–294
Price KR, Johnson IT, Fenwick GR (1987) The chemistry and
biological significance of saponins in food and feedstuffs.
CRC Crit Food Sci Nutr 26:27–135
Quazi HM (1975) Effect of cultivar and season on the con-
centration of saponins in lucerne (Medicago sativa L.).
NZ J Agric Res 18:227–232
Raizada RB, Srivastava MK, Kaushal RA, Singh RP
(2001) Azadirachtin, a neem biopesticide: Subchronic
toxicity assessment in rats. Food Chem Toxicol 39:
477–483
Rao AV, Sung MK (1995) Saponins as anticarcinogens. J Nutr
125:117s–124s
Regulation EC No 396/2005 of the European Parliament and of
the Council of Europe. http://eur-lex.europa.eu/
Regulation EC No 33/2008 of the European Parliament and of
the Council of Europe. http://eur-lex.europa.eu/
Regulation EC No 1095/2007 of the European Parliament and
of the Council of Europe. http://eur-lex.europa.eu/
San Martin R (2004) Use of Quillaja saponins to control
nematodes. Development of a commercial product: QL
AGRI. Abstract Book, International Conference on
Saponins, Pulawy, Poland, p 6
San Martin R, Magnunacelaya JC (2005) Control of plant-
parasitic nematodes with extracts of Quillaja saponaria.
Nematol 7:577–585
Saniewska A, Jurzysta M, Bialy Z (2001) Differential anti-
fungal activity of alfalfa (Medicago sativa L.) saponins
originated from roots and aerial parts of some ornamental
plant pathogens. Acta Agrobot 54:31–43
Saniewska A, Jurzysta M, Bialy Z (2003) The effect of alfalfa
(Medicago sativa) saponins on Botrytis tulipae and Phomanarcissi growth. Phytopathol Polon 27:15–27
Phytochem Rev
123984
Shakil NA, Pankaj KumarJ, Pandey RK, Saxena DB (2008)
Nematicidal prenylated flavanones from Phyllantus niruri.Phytochemistry 69:759–764
Small E, Jomphe M (1989) A synopsis of the genus Medicago(Leguminosae). Can J Bot 67:3260–3294
Spiegel Y, McClure MA (1995) The surface coat of plant-
parasitic nematodes: chemical composition, origin, and
biological role—a review. J Nematol 27:127–134
Sprag SG, Light ME, van Staden J (2004) Biological activities
and distribution of plant saponins. J Ethnopharm 94:
219–243
Tava A, Avato P (2006) Chemical and Biological activity of
triterpene saponins from Medicago species. NPC 1:
1159–1180
Tava A, Odoardi M (1996) Saponins from Medicago spp.:
chemical characterization and biological activity against
insects. In: Waller GR, Yamasaki K (eds) Advances in
experimental medicine and biology. Saponis used in
food and agriculture, vol 405. Plenum Press, New York,
pp 97–109
Tava A, Pecetti L (1998) Hemolytic activity and saponin
content in lucerne (Medicago sativa complex) genotypes.
J Gen Breed 52:33–37
Tava A, Oleszek W, Jurzysta M, Berardo N, Odoardi M (1993)
Alfalfa saponins and sapogenins: isolation and quantifi-
cation in two different cultivars. Phytochem Anal 4:
269–274
Tava A, Odoardi M, Oleszek W (1999) Seasonal changes of
saponin content in five alfalfa (Medicago sativa) cultivars.
Agric Mediterr 129:111–116
Tava A, Mella M, Bialy Z, Jurzysta M (2003) Stability of
saponins in alcoholic solutions: ester formation as arti-
facts. J Agric Food Chem 51:1797–1800
Tava A, Mella M, Avato P, Argentieri MP, Bialy Z, Jurzysta M
(2005) Triterpene saponins from leaves of Medicago ar-borea L. J Agric Food Chem 53:9954–9965
Tava A, Mella M, Avato P, Biazzi E, Pecetti L, Bialy Z, Jur-
zysta M (2009) New triterpenic saponins from the aerial
parts of Medicago arabica (L) Huds. J Agric Food Chem
57:2826–2835
Timbekova AE, Abubakirov NK (1984) Triterpene glycosides
from alfalfa. I. Medicoside G. A novel bidesmoside from
Medicago sativa. Khim Prirod Soedin 451–458
Timbekova AE, Abubakirov NK (1985) Triterpene glycosides
from alfalfa. II. Medicoside C. Khim Prirod Soedin
805–808
Timbekova AE, Abubakirov NK (1986a) Triterpene glycosides
from alfalfa. VII. Medicoside I. Khim Prirod Soedin
607–610
Timbekova AE, Abubakirov NK (1986b) Triterpene glycosides
of alfalfa. IV. Medicoside J. Khim Prirod Soedin 610–613
Timbekova AE, Larin MF, Yagudaev MR, Abubakirov NK
(1989) Triterpene glycosides of alfalfa. V. Medicoside H.
Khim Prirod Soedin 673–677
Timbekova AE, Verechagin AL, Semenov AA, Abubakirov
NK (1990) Triterpene glycosides of alfalfa. VI. Medico-
side L. Khim Prirod Soedin 221–227
Timbekova AE, Shashkov AS, Abubakirov NK (1993) Triter-
pene glycosides from alfalfa. VII. Medicosides E and F.
Khim Prirod Soedin 701–705
Timbekova AE, Isaev MI, Abubakirov NK (1996) Chemistry
and biological activity of triterpenoid glycosides from
Medicago sativa. In: Waller GR, Yamasaky K (eds)
Advances in experimental medicine and biology. Sapo-
nins used in food and agriculture, vol 405. Plenum Press,
New York, pp 171–182
Uri ND (1998) Development and use of biopesticides: impli-
cations of government policy and consumers’ preferences.
Technol Forecas Soc Change 59:291–304
Walker GE (2007) Effects of organic amendments, fertilizers
and fenamiphos on parasitic and free living nematodes,
tomato growth and yield. Nematol Med 35:131–136
Waller GR (1989) Biochemical frontiers of allelopathy. Biol
Plant 31:418–447
Waller GR, Jurzysta M, Thorne RLZ (1993) Allelopathic
activity of root saponins from alfalfa (Medicago sativa L.)
on weeds and wheat. Bot Bull Acad Sin 34:1–11
Waller GR, Jurzysta M, Thorne RLZ (1995) Root saponins
from alfalfa (Medicago sativa L.) and their allelopathic
activity on weeds and wheat. Allelopathy J 2:21–30
Zasada IA, Ferris H (2004) Nematode suppression with bras-
sicaceous amendments: application based upon glucosin-
olate profiles. Soil Biol Biochem 36:1017–1024
Zehavi U, Polacheck I (1996) Saponins as antimycotic agents:
glycosides of medicagenic acid. In: Waller GR, Yamasaki
K (eds) Advances in experimental medicine and biology.
Saponis used in traditional and modern medicine, vol 404.
Plenum Press, New York, pp 535–546
Phytochem Rev
123985