Download Identification of Trigonella accessions which lack antimicrobial

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SHORTCOMMUMCATION
Identification of Trigonella accessions which lack
antimicrobial activity and are suitable for forage development
J. E. Thomas 1 , S. K. Basu 1 , and S. N. Acharya 2
1 Department
of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, Canada T1 K 3M4 (e-mail:
[email protected]); and 2Agriculture and Agri-Food Canada, Lethbridge Research Centre, P.D. Box 3000,
Lethbridge, Alberta, Canada T1J 4B1. Received 10 August 2005, accepted 7 February 2006.
Thomas, J. E., Basu, S. K. and Acharya, S. N. 2006. Identification of Trigonella accessions which lack antimicrobial activity
and are suitable for forage development. Can. J. Plant Sci. 86: 727-732. Separate aqueous and ethanol extracts were prepared
from the leaves and seed of 75 different Trigonella accessions and their ability to inhibit the growth of enteric bacteria and yeast
was examined. No evidence of growth inhibition was observed, indicating that the Trigonella lines tested are suitable for forage
development and animal consumption.
Key words: Fenugreek, forage, antimicrobials, genetic diversity
Thomas, J. E., Basu, S. K. et Acharya, S. N. 2006. Identification des obtentions de Trigonella sans activite antibacterienne
utilisables pour la production fourragere. Can. J. Plant Sci. 86: 727-732. Les auteurs ont pris des feuilles et des semences de
75 obtentions de Trigonella pour en tirer des extraits aqueux et a l'ethanol. Ensuite, ils les ont examines pour voir s'ils inhibaient
la croissance des bacteries et des levures enteriques. Les auteurs n'ont releve aucune preuve d'activite antimicrobienne, signe que
les lignees testees conviennent a la production de fourrages pour la consommation des animaux.
Mots cles: Fenugrec, fourrage, antibacterien, diversite genetique
bility and chemical constituents; e.g., in saponin, fiber, pro­
tein, amino acid and fatty acid content in seed. High levels
of antimicrobial compounds within the leaves and/or stems
of forage cultivars could kill ruminant bacteria and yeast,
and could severely compromise the efficiency of fenugreek
forage to support weight gain in livestock and maximum
returns to the livestock industry. By contrast, the nutraceuti­
cal industry is under increasing pressure to provide product
labeling reflective of the medicinal properties and active
ingredients contained in its products. Genetic variability
must play a significant role in the commercialization of
fenugreek either as a medicinal herb or as a forage crop.
High quality and consistency in plant and seed properties
are important to product efficacy, as well as for the market­
ing strategies within these industries.
In this study, we examined the effect of aqueous and
ethanol extracts from the leaves and seed of 75 different
world accessions of Trigonella on the growth of some com­
mon enteric bacteria (Escherichia coli ATCC 25922 and
Escherichia coli DH5a, gram negative bacteria, and
Enterococcus jaecalis, a gram positive bacterium), and
some common yeast (Saccharomyces cerevisiae and
Saccharomyces bayanus) in order to assess their suitability
for use by the livestock industry as an alternative forage, or
as alternative antimicrobials for the nutraceutical industry.
Source of the Trigonella seed, the Accession Number (CN)
if known, and origin of the seed is identified in Table 1. Seeds
were planted in early May 2004 on irrigated land at the
Agriculture and Agri-Food Canada Research Centre at
Lethbridge (altitude 970 m), Alberta. The experimental plots
Fenugreek (Trigonellajoenum graecum L.) is an annual, self­
pollinating legume grown mainly as a spice, but it is also rec­
ognized for its medicinal and nutraceutical properties
(Sauvaire et al. 1996; Basch et al. 2003). However, the species
name "joenum-graecum" means "Greek hay" indicating its
use as a forage crop in the past (Petropoulos 2002). Fenugreek
is a dryland crop that responds well to minimalleve1s of irri­
gation (Mir et al. 1993; Moyer et al. 2003). Interest in culti­
vating fenugreek in temperate climates, such as that found in
western Canada, has increased because of its dryland adapta­
tion, high nutritive value irrespective of maturity (Mir et al.
1997) and the release of the first North American forage culti­
var Tristar. At present 500 ha of fenugreek are grown in west­
ern Canada to supply seed to the condiment market. However,
when Tristar seed is made available for forage production in
2007, this acreage is expected to grow rapidly.
Fenugreek plants and seed contain a complex array of
steroid saponins, flavonoids, and galactomannans which
form gum and oils (Sauvaire et al. 1996). Bhatti et al. (1996)
found that some sources of fenugreek seed possess wide­
spread antimicrobial activity against both gram negative and
gram positive bacteria, while extracts from fenugreek seed
examined by De et al. (1999) were unable to prevent growth
of either bacteria or yeast. Fenugreek seeds currently used
commercially have not been subjected to intensive selection
through modem breeding programs, and accessions collect­
ed from diverse sources express a high level of variability
among genotypes (Taylor et al. 1997, 2002; Huang and
Liang 2000; Moyer et al. 2003). These genotypes differ in
morphology, growth habit, biomass, seed production capa727
728
CANADIAN JOURNAL OF PLANT SCIENCE
Table 1. The Trigonelfa world accessions, their corresponding sources, and origin. Seed used in this study are identified by line or name given to the
seed in our collection, source of the seed, Accession Number (eN) if known, and origin of the seed.
Line
Source z
9095
AMBER
FI8
PGRC
AAFC - Lethbridge
CDC South
U of Saskatchewan
CDC South
U of Saskatchewan
CDC South
U of Saskatchewan
CDC South
U of Saskatchewan
AAFC - Lethbridge
India
India
PGRC
China
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
Unknown
Gujarat
Hyderabad
Chennai
Rajasthan
Lucknow
New Delhi
Guwahati
Amritsar
Madhya Pradesh
Bangalore
Kolkata
Mumbai
Bhubaneswar
Rajasthan
Amritsar
New Delhi
Kolkata
Gujarat
Hyderabad
Mumbai,
Varanasi
Lucknow
Pusbkar
Bhopal
Chennai
Imphal
Guwahati
Bangalore
Bhubaneshwar
Srinagar
Rajasthan
Rajasthan
Grocery store - Edmonton
CDC - North
PGRC
PGRC
F70
F80
F86
L3068
L3172
L3177
L3312
L3375
L367 I
L3672
L3673*
L3674*
L3675
L3676
L3677
L3678
L3679
L3680
L3681
L3682
L3683
L3684
L3685
L3689
L3690
L369 I
L3692
L3693
L3694
L3695
L3696
L3697
L3698
L3699
L3700
L3701
L3702
L3703
L3704
L3705
L3706
L3707
L3708
L3709
L3710
L3711
L3712
L3713
L3714
L3715
L3716
L3717
L3718
L3719
L3720
L3721
NGC 2001
PI 229626
PI 138687
PI 143504
CN number
OriginY
CN 19121
CDC Saskatchewan, Canada
Eston Saskatchewan, Canada
CN 19123
Uttar Pradesh, India
CN 19062
Hamadan, Iran
CN 19129
CN 19130
Washington, United States of America
Germany
CN 19132
CN19133
CN 19135
CN 19136
CN 19137
CN 19138
CN 19139
CN 19150
CN 19151
CN 19069
CN 19070
CN 19071
United Kingdom
Vienna, Austria
Poland
Germany
Germany
United Kingdom
Geneva, Switzerland
Romania
France
CDC Saskatchewan, Canada
CDC Saskatchewan, Canada
CDC Saskatchewan, Canada
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
India
CN 19118
CN 19062
Shiraz, Iran
Hamadan, Iran
THOMAS ET AL. -
FENUGEEK LACKING IN ANTIMICROBIAL ACTIVITY
729
Table 1. Continued
PI 195691
PI 199264
PI 211636
PI 269994
PI 577711
PI 577713
QUATRO
TRISTAR
X92-23-3
ZT-5
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
PGRC
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
19063
19064
19065
19066
19067
19068
19069
19118
19071
19070
Ethiopia
Greece
Afghanistan
Pakistan
~eknes,~orocco
~adrid,
Spain
CDC Saskatchewan, Canada
Shiraz, Iran
CDC Saskatchewan, Canada
CDC Saskatchewan, Canada
All the lines used were Trigonellafoenum-graecum L., with the exception of lines L3673* and L3674*, which were T. caerulea. All seed tested for antimi­
crobial properties was grown at Lethbridge, AB.
'Source: identifies where the seed was purchased; i.e., from suppliers within specific regions of a country (identified by region), from local markets (identi­
fied by city), and from crop development centers at AAFC - Agriculture and Agri-Food Canada, Lethbridge, AB; at CDC - Crop Development Centre, South,
University of Saskatchewan, Saskatoon, SK; and at PGRC - Plant Gene Resources of Canada, Saskatoon, SK.
YOrigin: identifies where seed varieties were developed or subjected to breeding selection as indicated by the supplier.
Fig. 1. A "representative sample" agar plate showing bacterial growth in the presence of Trigonella extracts and a positive control antibiot­
ic disc. E. coli ATCC 25922 was plated onto Mueller-Hinton agar and antibiotic discs impregnated with leaf extracts from fenugreek lines
L3673, PI 577713, F80 and TRISTAR were placed on the media along with a positive control containing 50 Ilg of streptomycin (S) to deter­
mine if they were able to inhibit bacterial growth.
were located in Orthic Dark Brown Chernozemic soil at N
49°42.009' and W 112°45.745'. Plots consisting of 120 seeds
were planted in 3-m-long rows using a custom built forage
seeder.
Plant extracts were prepared from 3 g of dry seed or 5 g
of mature fresh leaves. Plant materials were washed in ster­
ile distilled water, surface sterilized in a solution of 0.1 %
mercuric chloride (HgCl z) for 1 min and then rinsed four
times in sterile distilled water to remove any trace of the
mercuric chloride. Leaf materials were immediately crushed
in a sterile mortar and pestle, while seeds were first soaked
in 70% ethanol (2 mL per six seeds) overnight at room tem­
perature and then drained and crushed in a sterile mortar and
pestle the following day. All extracts were prepared in either
1.0 mL of 70% ethanol or 1.0 mL of sterile distilled water.
Extracts were absorbed into sterile lO-mm-diameter blank
antibiotic discs (Fisher Scientific International) for 1 min,
and then stored at 4°C in sterile plastic petri plates until use.
730
CANADIAN JOURNAL OF PLANT SCIENCE
Escherichia coli A Tee 25922
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0.2
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A
C
Cij
u
;ij
C­
A:--'
0.15
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0.1
,,
o
0.05'
..R··.·· If"":
a­
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 1
-0.05
Time (h)
Enterococcus faecalis
0.4
r------------------i
0.35
l:- 0.3
.~
B
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.. - ... ~
0.25
··1I
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,f
021
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.!:! 0.151
8-
... -....
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I
T
:~l:~~~2S=~~~~~,:j
Time (h)
Saccharomyces bayanus
1.2 - r - - - - - - - - - - - - - - - - - - - - ,
c
.fE" ...... -
l:­
'iii
~ 0.8
Cij
u
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...----.. ---~
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O+----.,---,----,----,--,-----r---r----r--.-----,.--r----.,
2
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Time (h)
'----------------------------­
Fig. 2. Growth of bacteria and yeast treated with Trigonella leaf extracts in liquid culture. A 100 ~L sample of overnight culture from (A)
E. coli ATCC 25922, (B) E. faecalis or (C) S. bayanus was inoculated into 5 mL, triplicate cultures of either TSB (bacteria) or Czapex Dox
broth (yeast), supplemented with 0 (Control .---.) or 100 ~L (Treated .---.) of aqueous extract from Tristar fenugreek leaves, The opti­
cal density (OD = 540 nm) of the culture was read at hourly intervals using a GILFORD Stasar III Spectrophotometer, and the mean ± stan­
dard deviation plotted. Liquid cultures were grown at 37 ± 2°C in a LAB-LINE shaking incubator set at 125 rpm.
THOMAS ET AL. ­ FENUGEEK LACKING IN ANTIMICROBIAL ACTIVITY
731
For each Trigonella line examined, three replicates of
some fenugreek seed (Bhatti et al. 1996), none of the plants
each aqueous and ethanol extract (Table 1) were tested for
that we examined possessed this activity. However, Taylor
their ability to inhibit the growth of E. coli ATCC 25922 and
et al. (2002) have shown that chemical composition can vary
E.coli DH5a (gram negative bacteria), E. jaecalis (a gram
among different accessions of fenugreek originating from
positive bacterium) and some common yeast (S. cerevisiae
different countries of the world, as well as for plant lines
and S. bayanus). These bacteria and yeast are considered
grown at three different sites in western Canada over a 2-yr
representative of the bacteria and yeast commonly found
period. Variation in genetics and environmental response of
within the digestive tracts of most animals. Overnight cul­
plants to production of phytochemicals also has been shown
tures of bacteria initially were grown at 37°C in Tryptic Soy
for the Australian sweet lupin (Lupinus angustifolius), the
Broth (TSB), and then a 100-J.lL aliquot of bacterial suspen­
Nigerian cowpea [Vigna unguiculata (L.) Walp], the
sion was plated onto Mueller-Hinton agar plates. Yeast were
Hawaiian kava (Piper methysticum Forster), and Capsicum
grown overnight in Czapex Dox broth, and a 100-J.lL aliquot
annum L. (Ruiz et al. 1995; Oluwatosin 1999; Lebot et al.
of yeast suspension was plated onto Czapex Dox agar plates.
1999; Zewdie and Bosland 2000).
Test discs containing the plant extracts were aseptically
Processed food and nutraceutical industries which use plant
transferred to the plates, four discs per agar plate, incubated
additives such as fenugreek to enhance product value are
at 37°C, and then examined for a zone of clearing surround­
responsible for providing labeling on their produce which
ing the discs at 24, 48 and 72 h.
reflects constituent contents and, addresses claims associated
To determine if soluble extracts from Trigonella were
with active compounds found in their products. It is important
able to prevent growth of bacteria or yeast in liquid culture,
to the economic development of these industries that genetic
100 J.lL each of E. coli ATCC 25922, E. jaecalis, S. cere­
and ecotype variations in plants used by these industries are
visiae and S. bayanus overnight liquid cultures were inocu­
properly characterized, and that new plant lines with associat­
lated into 5 mL, triplicate samples of either TSB (bacteria)
ed agronomic management packages capable of consistent
or Czapex Dox broth (yeast), supplemented with 0, 20, 40,
production of important constituents are developed. Industrial
60, 80 or 100 J.lL of aqueous extract from 5 g of Tristar fenu­
products marketed as antimicrobials must be developed from
greek leaves. Cultures were grown on a shaking platform in
plants showing genetic potential for antimicrobial activity.
liquid broth (37°C) and examined at 24, 48 and 72 h.
Our study indicates that under typical growth conditions used
None of the Trigonella leaf or seed extracts examined
in western Canada, the 75 plant accessions examined lack any
produced a zone of clearing around either the aqueous or
apparent antimicrobial activity, but are good candidates for
ethanol extract-soaked discs that were tested (Fig. 1).
further forage development. This study represents the most
Similarly, no inhibition of growth was observed either for
extensive survey of antimicrobial activity in Trigonella done
the bacteria or for the yeast cultures tested. Further, when
to date.
100 J.lL each of E. coli ATCC 25922, E. jaecalis, and S.
bayanus overnight liquid cultures were inoculated into 5
The authors gratefully acknowledge the Alberta Agriculture
mL, triplicate samples of either TSB (bacteria) or Czapex
Research Institute Farming for the Future Direct Funding Program,
Dox broth (yeast), supplemented with 0 (Control) or 100 J.lL
Agriculture and Agri-Food Canada Lethbridge Research Centre,
and the University of Lethbridge for support of a Graduate Student,
(Treated) of aqueous extract from Tristar fenugreek leaves
Saikat Basu and this research.
and examined at hourly intervals, all exhibited some stimu­
lation of growth (Fig. 2). No evidence of growth inhibition
Basch, E., Ulbricht, C., Kuo, G., Szapary, P. and Smith, M.
was observed. This indicated that all accessions could be
2003. Therapeutic applications of fenugreek. Altern. Med. Rev. 8:
used in the development of cultivars for forage purposes.
20-27.
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trum antibacterial activity against gram negative and gram
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chased in Pakistan was extracted into either water or
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Huang, W. Z. and Liang, X. 2000. Determination of two flavone
vented bacterial growth in zones surrounding each disc.
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Zones cleared of bacterial growth ranged in size from 12 to
Lebot, V., Johnston, E., Zheng, Q. Y., McKern, D. and
21 mm, and exhibited a direct dose response relationship
McKenna, D. J. 1999. Morphological, phytochemical, and genet­
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ic
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732
CANADIAN JOURNAL OF PLANT SCIENCE
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