Inhibition of potato cyst nematode hatch by lignans from ... - CSIC

INHmITION OF POTATO CYST NEMATODE HATCH
BY LIGNANS FROM Bupleurum salicifolium
(UNBELLIFERAE).
JOSE A. GONZÁLEZ, ANA ESTEVEZ-BRAUN,
RAFAEL ESTEVEZ-REYES, * and ANGEL G. RA VELO
Centro de Productos Naturales Orgánicos "Antonio González"
Instituto Universitario de Bio-orgánica, UniversitkuJ de La Laguna
C/Astrofisico Francisco Sánchez 2
38206 La Laguna, Tenerife, Spain
(Received June 28, 1993; accepted October 18, 1993)
Abstract-A series of lignans from Bupleurum salicifolium Soland (Umbelliferae)
were tesled for nemalostatic activity on the cysts and freed secondstage
juveniles of ¡he potalo cyst nematodes Globodera rostochiensis and G.
pallida. None of Ihe six lignans lested-bursehemin, rnatairesinol, syringaresinol,
Ihe novel produCI buplerol, guayarol. and a derivative, nortrachelogenin
triacetale-showed nemalicidal activity in an in vilro analysis wilh
second-stage juveniles, but significant differences were noled when Ihe Iignans
were assayed for nematostatic activity as cysl hatching inhibitors. Bursehemin
and matairesinol showed Ihe greatest activity, at concentrations of 50 ppm.
This is Ihe first known instance of a natural product inhibiting Ihe hatch of
Ihe nematode G. pallida. The HID (hatching inhibiting dosel of bursehemin
was estimated, and some conc\usions were drawn about the structure-activity
relationships of the Iignans under study.
Key Words-Potato cyst nernatodes, Globodera rostochiensis, G. pallida,
hatching inhibitors, lignans; Umbelliferae, Bupleurum salicifolium.
INTRODUCTION
Globodera rostochiensis and G. pallida, two cyst-fonning nematodes, are major
potato pests (Winslow and WilIis, 1972). They are high1y specialized pathogens
chamcterized by a narrow host mnge in the solanaceous plants (including potatoes,
tomatoes, aubergines, and tobacco). The potato cyst nematodes have CQ-
*To whorn correspondence should be addressed.
517

evolved with their major host, the potato, in their original habitat, the American
Andes (Stone, 1985), where they show broad genetic heterogeneity. Successful
survival strategies by this parasite involve mechanisms that permit the activation
of the infective unit on1y when the probability of finding a suitable host is high.
Eggs from Globodera spp. can remain viable for up to 20 years in the soil
(Winslow and WiIlis, 1972) and are induced to hatch by exudates from the
potato roots (Atkinson et al., 1987).
The diseovery of molecules regulating sorne stage of the life-eycle of the
parasite could provide control strategies other than the search for resistant phenotypes,
whieh is expensive and short-term (given the high variability of the
populations even within Europe), and the use of nematicides, which are among
the least environmentally aceeptable of all the crop-proteetion chemicals in use.
These substances may also be used for research into the processes of hatching
and diapause of eyst nematodes (Forrest and Farrer, 1983).
Triffitt (1930) and EHenby (1945) found that mustard root diffusate had a
neutralizing effect on the hatching of G. rostoehiensis, and Miigi (1970) reported
that root diffusates from several UmbeHiferae (Arehangeliea litoralis, Heracleum
sibirieum, Anthriseus silvestris, Pimpinella saxifraga, and P. major)
inhibited G. rostoehiensis hatch by 36-56% compared to eontrols. Asparagusic
acid (l,2-dithiolane-4-earboxylic acid) from Asparagus spp. (Liliaceae) has been
identified as a hatehing inhibitor of the eysts of G. rostoehiensis and Heterodera
glycines at adose of 50 ppm and 25°C (Takasugi et al., 1975).
When G. rostoehiensis and the tomato brown root rot complex (gray sterile
fungus when cultured, GSF) were both present in son, it was observed that the
population of nematode pathogens remained low (Graham, 1966). Glyn (1966)
found that this fungus produced exudates in malt extraet broth culture medium
that significantly inhibited the hatch of G. rostoehiensis (ca. 25% ofthe control).
The same principie as MeParland (1970) observed inhibiting the hatch of G.
rostoehiensis from exudates of the fungus Rhizoctonia solani is probably
invoIved.
Bupleurum salicifolium is endemic to the westem Canary Islands from Gran
Canaria to El Hierro. It is frequentIy found on cliffs up to 1000 m aboye sea
level (Bramwell and Bramwell, 1974). The pIant is rieh in shikimic pathway
derivatives (lignans, eoumarins, ftavonols, etc.). Other speeies ofthe genus have
been studied, and Muckensturm et al. (1982) reeorded the bioIogical activity of
a phenylpropanoid isolated from B. frutieosum that proved to be a strong antifeedant
for the inseet Mythimna unipunetata.
METHODS AND MATERIALS
Extraetion and Characterization of Lignans from B. salieifolium.
Leaves of wild specimens of B. salieifoLium (3.2 kg) colleeted in the Barranco
Rio Badajoz, Güimar (Tenerife) were extracted in a Soxhlet with EtOH.
After reduction of the ethanoI extraet by distillation in vacuo, the extract was

treated with petroleum ether and benzene, leaving a dark semisolid residue
(112.8 g), which was chromatographed repeatedly on silica gel and Sephadex
LH-20, yielding the following products: the triterpene betulin (0.4 g); coumarins
6,7,8-trimethoxy coumarin (6 mg), hemiarin (5 mg), and escopoletin (4 mg); a
polyacetylene (150 mg); and the lignans bursehemin (1) (93 mg), matairesinol
dimethyl ether (500 mg), buplerol (5) (43 mg), matairesinol (2) (47.4 mg),
nortrachelogenin (31.8 mg), guayarol (3) (lO mg), and syringaresinol (4) (15
mg).
Buplerol and guayarol were described for the first time by González et al.
(1990a). AH the compounds isolated were fuUy characterized using spectroscopic
techniques (UV, IR, IH NMR, 13C NMR, HMBC, HMQC, and MS)
(González et al., 1990b,c).
Nortrachelogenin triacetate (6) was obtained from nortrachelogenin in the
usual way. The structures of the lignans are shown in Figure 1.
Selection of Lignans. To investigate the inftuence of the lactone system on
hatching, a furofuranic type lignan, 4, was chosen. Compound 6 was used to
study the effect of hydroxy sterification of the aromatic group and the effect of
different groups in the B aromatic ring was studied using lignans 1, 2, 3, and
5.
Namatodes. The potato cyst nematodes were physiologically similar, collected
from potato fields on Tenerife and cultured in cultivar Désiré under glasshouse
conditions (20°C and 16 hr daylight). On June 20, 1991, the cysts were
Me~~co
Me
H
Me
B
O
0-1
Su_hemin 111
OH
Malaln .. linol (2)
Supleral (5)
OR
Nortrachelogenln R=H
M
M
OH
Guayaral (3)
X/OH
~~
~
H--. ·-H
O
Me
Nortrachelogenln Tria cela .. R=OAc (S)
FIG. 1. Structures of the lignans studied.
H ~ Syrlngar •• 'nol (4)

ecovered from the substrate (1: 3 sand/loan mixture) using a Schuiling centrifuge
(Hietbrink and Ritter, 1982) and stored in the dark at 4°e until used.
A 50 : 50 mixture of G. palUda pathotype Pa2/3 and G. rostochiensis pathotype
Rol (Phillips and Trudgill, 1983) was used in all experiments. The cysts
were not in their dormancy phase.
Bioassays for nematostatic Activity against Globodera spp. To test the
nematicidal activity of the lignans, batches of up to 1000 cysts were soaked
ovemight in distilled water and induced to hatch using a 10 mM ZnS04 solution
(Robinson and Nea1, 1959; elarke and Shepherd, 1966). Aliquots (lOO JLI)
containing approximately 50 two-day-old juveniles (second-stage juveniles, J2)
were placed in a water solution containing 250 ppm of the lignan and appropriate
controls were seto After 24 hr, percent mortality was recorded.
Nematostatic activity was investigated by studying the effect of the lignans
on cyst hatching. Batches of 10 cysts were soaked in distilled water for two
days, and the water was then replaced by the test solution containing 50 ppm
of the lignans and 10 mM ZnS04 as hatching agent. The total volume of the
assay was 2 cc.
Hatched juveniles were counted regularly, the cysts washed thoroughly
with distilled water and the test solution replaced with fresh stock. Each treatment
was applied to four replicates (except in the case of 4, when only 2
replicates were available) and the controls, one (el) with distilled water plus
0.5% EtOH and the other (e2) with the hatching agent plus 0.5% EtOH, the
solvent used with the lignans.
The experimental design was a randomized complete block and the results
were analyzed by a two-way ANOV A. To avoid zero values, one unit was
added to all the replicates. After examination of the data, a log transformation
was needed to normalize the data. The mean number of hatched juveniles at the
end of the experiment was compared using Duncan' s multiple range test at 5 %
significance leve!.
RESULTS AND DISCUSSION
Nematicidal Effect of Lignans. No nematicidal effect was apparent for doses
of up to 250 ppm (Table 1).
Nematostatic Effect of Lignans. The chronological hatch of cysts influenced
by the different lignans is shown in Figure 2. The analysis of variance showed
highly significant differences (P < 0.01) between treatments, time, and the
interaction treatments x time.
Duncan's multiple range test clustered the means in nonsignificant groups
(Figure 3). After 14 days, all the treatments hatched more juveniles than were
hatched in distilled water (el). The number of juveniles hatched when lignans

TABLE 1. PERCENT MORTAUTY OP TWO-DAY-OLD Globodera sp. SECOND STAGE
JUVENILES APTER 24-h EXPOSURE TO 250 ppm OP LIGNANS
Lignan added
None
Matairesinol (2)
Bursehemin (1)
Nortrachelogenin triacetate (6)
Guayarol (3)
Syringaresinol (4)
Bupleral (5)
Mortality (%)
30.6
20
23.1
24.2
25.9
31.3
34.5
400
N
3-
.

1 or 2 were present was significantly different from the control (C2) containing
10 mM ZnS04 as hatching agent, the hatch being reduced by 69.96% (1) and
55.14% (2). The behavior of lignan 1 also díffered significantly from that of
molecules 3 and 5, which have a similar structure.
The HIDso was estimated for lignan 1 using the best fitted curve (y =
-17.032 * lnx + 97.668) (Figure 4). Only 16.42 ppmofthe lignan was needed
to reduce hatch by 50% over a two week periodo
Examination of the juveniles hatched during the experiment showed that
there were no differences in the effect of lignans on G. pallida or G. rostochiensis,
and it would seem that the inhibitory hatching activíty of the lignans tested
is nonspecific. This is the first time that a natural product has been shown to
affect G. pallida hatching behavior.
Structure-Activity Relationship in Lignans. The presence of a methylenedíoxy
group in the dibenzyl-butyrolactone skeleton aromatíc ring B would seem
to playa part in the nematostatic activity of the products tested. Lignans 3 and
5 have the same skeleton as 1 except in this ring where the methylene-díoxy
group is replaced by a methoxy and a hydroxyl in 5 or two hydroxyls in 3.
These changes significantly reduced nematostatic activity.
In compounds with no methylene-dioxy group, activity increased according
to the number of free hydroxy groups. Thus lignans 2 and 3, which have two
free OH groups, showed more nematostatic actívity than 5, which has only one,
while 6, which has no free OH, displayed the least nematostatic activíty of all,
signíficantly dífferent from that of 2 and 3.
Apart from the absence of free hydroxyls, lignan 6 also has a relatively
volumínous acetate group at posítíon 2 in the lactone ring, which may partIy
figure as a possible steric bulk between this compound and the hypothetical
100
\ / y=97.668·17.032"ln x
¡R l .098i
80 \
.,
¿
\
'O 60
'- /
.c: ...............
.-.. .
~
: .....
J: 40
'"-,
--
20
HID .. =16A2 ppm
o o 10 20 30 40 50
[Bursehernin] (ppm)
FIG. 4. Reduction in potato cyst nematode hatch by bursehemin and hatching inhibiting
dose (HID so ).

eceptor on the nematode eggshell suggested by Atkinson and Taylor (1980,
1983).
Four of the above lignans have also been isolated in considerable quantities
from the ethanol root extracts of B. salicifolium (27.5 g): 4,5-matairesinol (2)
(13.1 mg), matairesinol dimethyl ether (15 mg), bursehernin (1) (23 mg), and
nortrachelogenin (6.3 mg) (Estévez-Reyes et al., 1992, 1993).
It is possible that sorne biogenetic compounds from the shikimic pathway,
lignans or polyphenols with similar structures, were involved in the inhibitory
effect recorded by Miigi (1970) when studying root extracts of umbelliferous
plants as cyst nematode hatching inhibitors.
Acknowledgments-The authors are grateful to the "Gobierno de La Comunidad Autónoma
Canaria" (Project No. 11/08-03-90) for financial help and for a postdoctoral grant to J .A. Gonzáles.
A. Estévez-Braun wishes 10 Ihank the "Ministerio de Educación y Ciencia" for a fellowship.
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