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Content uploaded by Valtcho D Jeliazkov (Zheljazkov)
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Herbicides for Weed Control in Blessed Thistle (Silybum marianum)
1
VALTCHO D. ZHELJAZKOV, IVAN ZHALNOV, and NEDKO K. NEDKOV
2
Abstract: Blessed thistle is an important medicinal crop in Europe and recently has become more
significant in North America. A limiting factor in blessed thistle production is weed interference.
Field experiments were conducted near Plovdiv, Bulgaria, to study the effect of selected herbicides
on weed control, crop productivity, and crop quality. Seed yields of blessed thistle were increased
with metribuzin alone at 0.5 kg ai/ha, pendimethalin alone 1.32 kg ai/ha, pendimethalin at
1.32 kg ai/ha plus metribuzin at 0.5 kg ai/ha, trifluralin at 0.84 kg ai/ha plus linuron at 1.0 kg ai/
ha, and in the hand-weeded control compared to the nonweeded control (nontreated check).
Pendimethalin and metribuzin were safe both alone and in combination for weed control in
blessed thistle. Bentazon at 0.96 kg ai/ha inhibited blessed thistle development and reduced seed
yields compared to the untreated check. Generally, weed control increased the content of
silymarin and decreased the amount of seed oil. Overall, seeds contained 0.26 to 0.36%taxifolin,
0.69 to 0.99%silydianin plus silycristin, 1.31 to 1.78%silybin, and 0.27 to 0.39 %isosilybin.
Nomenclature: Bentazon; linuron; metribuzin; pendimethalin; trifluralin; blessed thistle, Silybum
marianum (L.) Gaertn. ‘Silmar’
#
3
SLYMA.
Additional index words: Medicinal plants, seed oil, seed yield, silymarin, taxifolin, silydianin,
silycristin, silybin, isosilybin.
INTRODUCTION
Blessed thistle, also known as milk thistle, has
become one of the most widely grown and econom-
ically viable medicinal plants in Europe including
Bulgaria (Zheljazkov et al. 1996), and recently is
coming to prominence in North America. Blessed
thistle achenes (seeds) contain the biologically active
flavonolignan complex silymarin, that consists of
silybin, silydianin, and silycristin. In addition, the
fruits contain seed oil composed of mainly linoleic
and oleic acids. Silymarin from blessed thistle fruits is
used for the production of hepatoprotectors for the
treatment of liver diseases, mainly cirrhosis (Boerth
and Strong 2002; He et al. 2002; Jacobs et al. 2002).
Recently, blessed thistle extract was shown to possess
anticarcinogenic properties (Kohno et al. 2002) and
silybin was found to suppress cholesterol synthesis
(Nassuato et al. 1991). Several commercial medicines
and plant extracts containing blessed thistle extract
are being produced and offered under different trade
names in Europe and recently in the United States
and Canada (including Nova Scotia).
Blessed thistle is very sensitive to most of the
herbicides used in other crops (Cacso et al. 1977;
Topalov et al. 1983). Blessed thistle seeds have pappi
and are easily dispersed before harvest (Hecht et al.
1992; Todorov et al. 1990), resulting in blessed thistle
emerging as a weed in the next crop. In North
America, blessed thistle is a noxious weed in Oregon
(category B), Texas (category S2), and Washington
(category A) (Plant Protection and Quarantine 2002),
but not in any of the provinces in Canada (USDA-
ARS 2005). Blessed thistle is being grown as a crop in
Saskatchewan and in Atlantic Canada.
A limiting factor in blessed thistle production in
Bulgaria and in other parts of Europe has been lack
of weed control (Topalov et al. 1983). There are few
publications on weed control in blessed thistle, but
some authors recommend the use of PRE applications
of alachlor at 1.0 to 1.5 kg ai/ha, linuron at 1.0 kg ai/
ha, trifluralin at 0.48 to 0.72 kg/ha, and a combination
of alachlor at 1.5 kg ai/ha plus linuron at 1.0 kg/ha
1
Received for publication September 25, 2005, and in revised form May
4, 2006.
2
Research professor, Mississippi State University, North Mississippi
Research & Ext. Center, 5421 Hwy. 145 South, Verona, MS 38879;
Associate professor, Department of Farming, University of Agriculture,
12 Mendeleev str., 4000 Plovdiv, Bulgaria; Senior Researcher at
the Research Institute for Roses and Medicinal Plants, 49 Osvobo-
jdenia Blv, 6100, Kazanlak, Bulgaria. Corresponding author’s E-mail:
vj40@pss.msstate.edu.
3
Letters following this symbol are a WSSA-approved computer code
from Composite List of Weeds, Revised 1989. Available only on computer
disk from WSSA, 810 East 10th Street, Lawrence, KS 66044-8897.
Weed Technology. 2006. Volume 20:1030–1034
1030
(Topalov et al. 1983). In Hungary, Cacso et al. (1977)
tested 58 herbicides in the greenhouse and 19 PRE
and 13 POST herbicides in small-plot experiments
and found that blessed thistle was highly sensitive to
many herbicides. They reported that best weed
control in blessed thistle was achieved with PRE
applications of isopropalin at 1.6 kg ai/ha and benefin
at 0.09 to 0.1 kg ai/ha. However, these herbicides did
not provide 100%weed control. The objectives of this
study were to evaluate five of the more promising
herbicides for weed control and the effect of weed
control on blessed thistle yield and quality.
MATERIALS AND METHODS
Field experiments were conducted near Plovdiv,
Bulgaria, in 1995 and 1996 with the blessed thistle
cultivar ‘Silmar’, the most widely grown cultivar in the
country (Zheljazkov et al. 1996). The experiments were
conducted as a randomized complete block design with
3.0 by 10.0 m (30 m
2
) plots, in four replications. Treat-
ments evaluated were: (1) nontreated check (control
without weeding), (2) hand weeded (single hand
removal of the weeds), (3) trifluralin at 0.84 kg/ha
plus linuron at 1.0 kg/ha, (4) trifluralin at 0.84 kg/ha
plus bentazon at 0.96 kg/ha, (5) pendimethalin at
1.32 kg/ha, (6) metribuzin at 0.5 kg/ha, and (7) pendi-
methalin at 1.32 kg/ha plus metribuzin at 0.5 kg/ha.
Prior to planting, nitrogen (100 kg N/ha as NH
4
),
phosphorus (66 kg/ha as P
2
O
5
), and potassium
(83 kg/ha as K) were applied to the test site as
recommended for blessed thistle fertilization in the
region (Topalov et al. 1983) and based on the amount
of available P and K in the soil prior to seeding. The
soil was a calcareous alluvial-meadow soil (Koinov
1968) or calcaric fluvisols upon the FAO-UNESCO
(1974) classification of world soils. The sand, silt, and
clay contents of the soils were approximately 21, 41,
and 38%, respectively, making it a clay loam. The soil
was fall plowed to a 25- to 30-cm depth, and the next
spring tillage was done to 8- to 10-cm depth, 3 to 5 d
before seeding. In both years, blessed thistle was
seeded on March 12 at an interrow space of 45 cm
(Foldesi and Barsi 1983), a seeding rate of 15 kg/ha,
and at 2- to 3-cm depth (Topalov et al. 1983).
Herbicides were applied with a compressed air
backpack sprayer fitted with a flat-fan nozzle tips
4
calibrated to deliver 250 L water/ha at 200 kPa.
Trifluralin was applied PPI and incorporated 12 cm
deep by disking twice. Linuron, pendimethalin, and
metribuzin were applied PRE after seeding, and
bentazon was applied POST at the two- to five-leaf
stage of the weeds. Bentazon treatments included
Tween 20
5
at 0.25%v/v.
Specific composition and density of weeds was
established by counting the weeds from 2 m
2
in each
plot, just before harvest. Blessed thistle plants from the
whole plots, each 3.0 by 10.0 m, were harvested with
a swatter and left on the field for 3 to 4 d to allow all
the seeds to mature and dry. Subsequently, seeds were
threshed using a small plot research combine
6
and
dried to uniform 9%moisture. The extraction of active
compounds from blessed thistle seeds is usually a two-
step process; the first step is the extraction of the seed
oil (the lipids) and the second step is the extraction of
flavonolignan complex with organic solvents. Research
has demonstrated that prior removal of seed oil
(defattening) and higher extraction temperatures im-
prove the yields of silymarin from the seed meal
(defatted seeds) (Wallace et al. 2005). The seed oil
content was established by a 4-h extraction of the
achenes with hexane in a Soxhlet apparatus, with
a subsequent drying of the hexane extract at 105 C to
a constant weight. The content of silymarin was
obtained by acetone extraction for 12 h of the seed
meal, was conducted by a commercial company
7
using
its own method. The acetone in the extract was distilled
off under vacuum. The dry residue was dissolved in
methanol and brought up to 50 ml in a volumetric flask.
Five milliliters of this solution was diluted five times
with methanol (to 25 ml). Ten milliliters from the latter
was further diluted five times with methanol (to 50 ml).
The absorption of the resulting solution was measured
at a 288-nm wavelength and compared against a control
sample of 1-cm-thick methanol layer. The final percent-
age of silymarin was calculated using the formula:
Silymarin%~Asample|Bst:|12:5G
Ast:|Bsample
where:
A
sample
5extinction of the sample
A
st.
5extinction of the standard
B
sample
5weight of the sample
B
st.
5weight of the standard
5
Tween 20 (polyoxyethylenesorbitan monolaurate) from Agria, Plov-
div, Bulgaria.
6
Winterstieger Nurserymaster, Zentrale, Austria.
7
Bulgarska Rosa Sevtopolis, Kazanluk, Bulgaria.
4
Zavod za Selskostopanska Technica, Russe, Bulgaria.
WEED TECHNOLOGY
Volume 20, Issue 4 (October–December) 2006 1031
G5concentration of the internal
standard substance
The percentages of taxifolin, silydianin, silycristin,
silybin, and isosilybin were obtained using liquid
chromatography
8
with an UV detector and tetrahy-
drofuran modifier, using a mobile phase of 0.02 M
phosphate buffer and 0.05 M potassium nitrate in
35%tetrahydrofuran-water at pH 3.0 and run at
1.0 ml/min.
Data analyses were performed using two-way
ANOVA with SAS (SAS Institute 2000). Where the
main effects or interactions were found to be
significant, Duncan’s multiple range test was used to
separate means.
RESULTS AND DISCUSSION
Sixteen weed species, typical for the region, were
identified prior to harvest, of which 11 were annual
(including nine dicotyledonous and two monocotyle-
donous) and five perennial (including three dicotyle-
donous and two monocotyledonous). Of these, most
abundant were redroot pigweed (Amaranthus retro-
flexus L.),green foxtail [Setaria viridis (L.) Beauv.],
and bermudagrass [Cynodon dactylon (L.) Pers.]. The
other weeds species included the annual dicotyledon-
ous prostrate pigweed (Amaranthus blitoides S.Wats.),
common lambsquarters (Chenopodium album L.),
jimsonweed (Datura stramonium L.), wild buckwheat
(Polygonum convolvulus L.), prostrate knotweed
(Polygonum aviculare L.), black nightshade (Solanum
nigrum L.), common cocklebur (Xanthium strumarium
L.), velvetleaf (Abutilon theophrasti Medik.), and the
monocotyledonous large crabgrass (Digitaria sangui-
nalis L.). The perennials included dicotyledonous field
bindweed (Convolvulus arvensis L.), motherwort
(Leonurus cardiaca L.), and Canada thistle (Cirsium
arvense L.), and the monocotyledonous johnsongrass
[Sorghum halepense (L.) Pers.]. The annual weed
density at harvest varied significantly (Table 1) with
the highest weed infestation found in the nontreated
check. A single weeding at the three- to five-leaf stage
of weeds reduced weeds by 50%at the end of
vegetation period (i.e., prior to harvest). All herbicide
treatments significantly lowered weed density com-
pared to the nontreated check. The best control of
annual weeds was achieved with the herbicide
combinations of pendimethalin plus metribuzin for
dicots and trifluralin plus linuron for monocots,
indicating that linuron is more effective than benta-
zon for control of monocots. Perennial weed density
was not affected by the herbicides tested or by the
single hand removal of weeds, since the tested
herbicides are not active on established perennial
monocotyledonous and dicotyledonous weeds (Fet-
vadjieva et al. 1994; Mitchell and Abernethy 1993). At
harvest, annual weed infestation was lowest in the
metribuzin treatment, followed by pendimethalin and
pendimethalin plus metribuzin treatments; hence, the
herbicides pendimethalin and metribuzin could pro-
vide weed control in blessed thistle.
A single hand removal of the weeds gave a seed
yield increase of 27 and 40%in 1995 and 1996,
respectively, compared to the nontreated check
(Table 2). Differences in seed yields between the
nontreated check and the hand-weeded treatment
were significant and support previous reports that
blessed thistle plants develop and grow very quickly
and successfully suppress the weeds in later stages, but
plants are very sensitive to weed interference in their
initial stages of growth (Chiavari et al. 1991; Topalov
et al. 1983). Our results support those of Schunke
(1992) that if hand hoeing is done at this time (i.e., the
initial stages of blessed thistle growth), weed control
in blessed thistle crop would not be a significant
8
PYE Unicam 4100 liquid chromatograph.
Table 1. Effect of treatments on weed density in blessed thistle crops (pooled data for 1995 and 1996).
Treatments Rate
Annual weeds Perennial weeds
Monocots Dicots Monocots Dicots
kg ai/ha -------------------------------------------------------------------------------- n o . weeds/m
2
------------------------------------------------------------------------------
Nontreated check (without weeding) 32 a
a
41 a 4 b 1 b
Single hand weeding 23 b 19 b 3 b 1 b
Trifluralin plus linuron 0.84 +1.0 3 d 9 c 13 a 3 b
Trifluralin plus bentazon 0.84 +0.96 7 c 12 bc 3 b 7 a
Pendimethalin 1.32 6 cd 13 bc 0 0
Metribuzin 0.50 10 c 4 d 2 b 4 b
Pendimethalin plus metribuzin 1.32 +0.50 7 cd 4 d 5 b 3 b
a
Means with the same letter within a year (column) are not significantly different at P #0.05 using Duncan’s multiple range test.
ZHELJAZKOV ET AL.: WEED CONTROL IN SILYBUM MARIANUM L.
1032 Volume 20, Issue 4 (October–December) 2006
problem. All herbicide treatments except bentazon,
which exhibited phytotoxicity on blessed thistle, had
higher yields than the nontreated check (Table 2).
Pendimethalin and metribuzin treatments in combi-
nation increased yield by up to 25%over trifluralin
plus linuron treatment. These results suggest that the
herbicides pendimethalin and metribuzin can success-
fully be used either alone or in combination for weed
control in blessed thistle.
The seed oil content was highest in blessed thistle
seeds when no herbicides were used (in the nontreated
check and in the hand-weeded treatment, respectively)
in both years (Table 3). Pendimethalin or metribuzin
alone decreased seed oil content relative to the
nontreated check in both years, while trifluralin plus
bentazon decreased seed oil in 1995 but not in 1996.
Content of seed oil is important, since after extrac-
tion, blessed thistle seeds are used as feed for domestic
animals (Potkanski et al. 1991). The oil content in the
seeds from our experiment varied from 18.4 to 26.2%,
which is lower than that reported by Hamid et al.
(1983) and Omer et al. (1993) and might be due to
differences in the tested cultivars or to environmental
conditions.
Weed control by herbicides increased silymarin
content of seeds for all herbicide combinations but
less strongly when metribuzin was applied alone
(Table 3). Our data seem to suggest that the increase
in silymarin content in the herbicide treatments may
be due to the direct effect of herbicides on blessed
Table 2. Seed yield of blessed thistle in response to hand weeding and weed control with herbicides.
Treatments Rate
Seed yield Percent of check
1995 1996 1995 1996
kg ai/ha ---------------------------------------- kg/ha------------ ---------------------------- ----------------------------------------- %---------------------------------------
Nontreated check (no weeding) 655.3 c
a
737.5 c 100 100
Single hand weeding 832.5 b 1032.1 b 127 140
Trifluralin plus linuron 0.84 +1.0 1070.0 a 977.8 b 163 133
Trifluralin plus bentazon 0.84 +0.96 462.4 d 620.1 d 71 84
Pendimethalin 1.32 1080.3 a 1026.4 b 165 139
Metribuzin 0.50 1094.8 a 1141.0 ab 167 155
Pendimethalin plus metribuzin 1.32 +0.50 1133.2 a 1273.1 a 173 173
a
Means with the same letter within a year (column) are not significantly different at P #0.05 using Duncan’s multiple range test.
Table 3. Seed oil and silymarin content in seeds of blessed thistle in response to hand weeding and weed control with herbicides.
Treatments Rate
Seed oil Silymarin
1995 1996 1995 1996
kg ai/ha ------------------------------------------------------------------------------------------------- %-------------------------------------------------------------------------------------------------
Nontreated check (no weeding) 24.6 a
a
25.9 a 2.3 b 2.0 b
Single hand weeding 24.1 a 26.2 a 2.2 b 2.1 b
Trifluralin plus linuron 0.84 +1.0 22.8 ab 23.6 ab 2.7 a 3.0 a
Trifluralin plus bentazon 0.84 +0.96 20.8 b 22.1 ab 2.6 a 3.2 a
Pendimethalin 1.32 19.7 b 18.4 b 2.7 a 2.9 a
Metribuzin 0.50 19.5 b 20.2 b 2.5 ab 3.1 a
Pendimethalin plus metribuzin 1.32 +0.50 22.8 ab 21.8 ab 2.9 a 2.8 a
a
Means with the same letter within a year (column) are not significantly different at P #0.05 using Duncan’s multiple range test.
Table 4. Taxifolin, silydianin and silycristin, silybin, and isosilybin content in seeds of blessed thistle in response to hand weeding and weed control with
herbicides (average for 1995 and 1996).
Treatment Rate Taxifolin Silydianin +silycristin Silybin Isosilybin
kg ai/ha ------------------------------------------------------------------------------------------- %------------------------------------------------------------------------------------------
Nontreated check (no weeding) 0.36 0.99 1.78 0.39
Single hand weeding 0.27 0.75 1.39 0.30
Trifluralin plus linuron 0.84 +1.0 0.31 0.86 1.58 0.34
Trifluralin plus bentazon 0.84 +0.96 0.26 0.69 1.31 0.27
Pendimethalin 1.32 0.31 0.82 1.54 0.31
Metribuzin 0.50 0.31 0.84 1.49 0.31
Pendimethalin plus metribuzin 1.32 +0.50 0.27 0.70 1.36 0.28
WEED TECHNOLOGY
Volume 20, Issue 4 (October–December) 2006 1033
thistle plants, rather than to the removal of stress with
weeds being controlled. Indeed, previous research has
demonstrated that herbicides may increase plant
secondary metabolites synthesis and accumulation in
some other crops (Cosio et al. 1985; Ko¨mives and
Casida, 1996; Lydon and Duke, 1989; Zheljazkov and
Zhalnov, 1995).
Overall, seeds contained 0.26 to 0.36%taxifolin,
0.69 to 0.99%silydianin plus silycristin, 1.31 to 1.78%
silybin, and 0.27 to 0.39 %isosilybin (Table 4). Due
to the high variation within a treatment, there were no
differences in the content of taxifolin, silydianin plus
silycristin, silybin, and isosilybin between the treat-
ments at P #0.05. Silymarin content in the seeds
varied depending on the level of weed control. This
supports the studies of Cacso et al. (1977) of
significant variation in total flavonoids, induced by
weed control with some herbicides.
Our results suggest that weed control in blessed
thistle could be obtained by single weed removal at
initial stages of crop development or by using
herbicides pendimethalin and metribuzin either alone
or in combination.
ACKNOWLEDGMENTS
This work was partially supported by the Bulgarian
Ministry of Agriculture grant awarded to V. Zheljaz-
kov and I. Zhalnov, and by AgriFutures Nova Scotia
grant
#
190 awarded to V. D. Zheljazkov (Jeliazkov).
Authors thank Dr. Claude D. Caldwell and Ms.
Stephanie Butler from the Nova Scotia Agricul-
tural College for their helpful corrections and sugges-
tions.
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ZHELJAZKOV ET AL.: WEED CONTROL IN SILYBUM MARIANUM L.
1034 Volume 20, Issue 4 (October–December) 2006