Identification of Trigonella accessions which lack antimicrobial activity and are suitable for forage development

SHORT COMMUNICATION 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 T1K 3M4 (e-mail: thomas@uleth.ca); and 2 Agriculture and Agri-Food Canada, Lethbridge Research Centre, P.O. 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 activité antibactérienne utilisables pour la production fourragère. 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 à l éthanol. Ensuite, ils les ont examinés pour voir s ils inhibaient la croissance des bactéries et des levures entériques. Les auteurs n ont relevé aucune preuve d activité antimicrobienne, signe que les lignées testées conviennent à la production de fourrages pour la consommation des animaux. Mots clés: Fenugrec, fourrage, antibactérien, diversité génétique 727 Fenugreek (Trigonella foenum graecum L.) is an annual, selfpollinating legume grown mainly as a spice, but it is also recognized for its medicinal and nutraceutical properties (Sauvaire et al. 1996; Basch et al. 2003). However, the species name foenum-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 minimal levels of irrigation (Mir et al. 1993; Moyer et al. 2003). Interest in cultivating fenugreek in temperate climates, such as that found in western Canada, has increased because of its dryland adaptation, high nutritive value irrespective of maturity (Mir et al. 1997) and the release of the first North American forage cultivar Tristar. At present 500 ha of fenugreek are grown in western 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 widespread 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 modern breeding programs, and accessions collected 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 capability and chemical constituents; e.g., in saponin, fiber, protein, 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 nutraceutical 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 marketing 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 common enteric bacteria (Escherichia coli ATCC 25922 and Escherichia coli DH5α, gram negative bacteria, and Enterococcus faecalis, 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

728 CANADIAN JOURNAL OF PLANT SCIENCE ARTICLE IN PRESS Table 1. The Trigonella 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 (CN) if known, and origin of the seed. Line Source z CN number Origin y 9095 PGRC CDC Saskatchewan, Canada AMBER AAFC - Lethbridge CN 19121 Eston Saskatchewan, Canada F18 F70 F80 F86 L3068 AAFC - Lethbridge CN 19123 Uttar Pradesh, India L3172 India L3177 India L3312 PGRC CN 19062 Hamadan, Iran L3375 China L3671 PGRC CN 19129 Washington, United States of America L3672 PGRC CN 19130 Germany L3673* PGRC L3674* PGRC CN 19132 United Kingdom L3675 PGRC CN 19133 Vienna, Austria L3676 PGRC CN 19135 Poland L3677 PGRC CN 19136 Germany L3678 PGRC CN 19137 Germany L3679 PGRC CN 19138 United Kingdom L3680 PGRC CN 19139 Geneva, Switzerland L3681 PGRC CN 19150 Romania L3682 PGRC CN 19151 France L3683 PGRC CN 19069 CDC Saskatchewan, Canada L3684 PGRC CN 19070 CDC Saskatchewan, Canada L3685 PGRC CN 19071 CDC Saskatchewan, Canada L3689 Unknown India L3690 Gujarat India L3691 Hyderabad India L3692 Chennai India L3693 Rajasthan India L3694 Lucknow India L3695 New Delhi India L3696 Guwahati India L3697 Amritsar India L3698 Madhya Pradesh India L3699 Bangalore India L3700 Kolkata India L3701 Mumbai India L3702 Bhubaneswar India L3703 Rajasthan India L3704 Amritsar India L3705 New Delhi India L3706 Kolkata India L3707 Gujarat India L3708 Hyderabad India L3709 Mumbai, India L3710 Varanasi India L3711 Lucknow India L3712 Pushkar India L3713 Bhopal India L3714 Chennai India L3715 Imphal India L3716 Guwahati India L3717 Bangalore India L3718 Bhubaneshwar India L3719 Srinagar India L3720 Rajasthan India L3721 Rajasthan India NGC 2001 Grocery store - Edmonton PI 229626 CDC - North PI 138687 PGRC CN 19118 Shiraz, Iran PI 143504 PGRC CN 19062 Hamadan, Iran

ARTICLE IN PRESS THOMAS ET AL. FENUGEEK LACKING IN ANTIMICROBIAL ACTIVITY 729 Table 1. Continued PI 195691 PGRC CN 19063 Ethiopia PI 199264 PGRC CN 19064 Greece PI 211636 PGRC CN 19065 Afghanistan PI 269994 PGRC CN 19066 Pakistan PI 577711 PGRC CN 19067 Meknes, Morocco PI 577713 PGRC CN 19068 Madrid, Spain QUATRO PGRC CN 19069 CDC Saskatchewan, Canada TRISTAR PGRC CN 19118 Shiraz, Iran X92-23-3 PGRC CN 19071 CDC Saskatchewan, Canada ZT-5 PGRC CN 19070 CDC Saskatchewan, Canada All the lines used were Trigonella foenum-graecum L., with the exception of lines L3673* and L3674*, which were T. caerulea. All seed tested for antimicrobial properties was grown at Lethbridge, AB. z Source: identifies where the seed was purchased; i.e., from suppliers within specific regions of a country (identified by region), from local markets (identified 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. y Origin: 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 antibiotic 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 µg of streptomycin (S) to determine 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 sterile distilled water, surface sterilized in a solution of 0.1% mercuric chloride (HgCl 2 ) 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 temperature 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 10-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 ARTICLE IN PRESS Fig. 2. Growth of bacteria and yeast treated with Trigonella leaf extracts in liquid culture. A 100 µl 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 µl (Treated --- ) of aqueous extract from Tristar fenugreek leaves. The optical density (OD = 540 nm) of the culture was read at hourly intervals using a GILFORD Stasar III Spectrophotometer, and the mean ± standard deviation plotted. Liquid cultures were grown at 37 ± 2 C in a LAB-LINE shaking incubator set at 125 rpm.

ARTICLE IN PRESS THOMAS ET AL. FENUGEEK LACKING IN ANTIMICROBIAL ACTIVITY 731 For each Trigonella line examined, three replicates of each aqueous and ethanol extract (Table 1) were tested for their ability to inhibit the growth of E. coli ATCC 25922 and E.coli DH5α (gram negative bacteria), E. faecalis (a gram positive bacterium) and some common yeast (S. cerevisiae and S. bayanus). These bacteria and yeast are considered representative of the bacteria and yeast commonly found within the digestive tracts of most animals. Overnight cultures of bacteria initially were grown at 37 C in Tryptic Soy Broth (TSB), and then a 100-µL aliquot of bacterial suspension was plated onto Mueller-Hinton agar plates. Yeast were grown overnight in Czapex Dox broth, and a 100-µL aliquot of yeast suspension was plated onto Czapex Dox agar plates. Test discs containing the plant extracts were aseptically transferred to the plates, four discs per agar plate, incubated at 37 C, and then examined for a zone of clearing surrounding the discs at 24, 48 and 72 h. To determine if soluble extracts from Trigonella were able to prevent growth of bacteria or yeast in liquid culture, 100 µl each of E. coli ATCC 25922, E. faecalis, S. cerevisiae and S. bayanus overnight liquid cultures were inoculated into 5 ml, triplicate samples of either TSB (bacteria) or Czapex Dox broth (yeast), supplemented with 0, 20, 40, 60, 80 or 100 µl of aqueous extract from 5 g of Tristar fenugreek leaves. Cultures were grown on a shaking platform in liquid broth (37 C) and examined at 24, 48 and 72 h. None of the Trigonella leaf or seed extracts examined produced a zone of clearing around either the aqueous or ethanol extract-soaked discs that were tested (Fig. 1). Similarly, no inhibition of growth was observed either for the bacteria or for the yeast cultures tested. Further, when 100 µl each of E. coli ATCC 25922, E. faecalis, and S. bayanus overnight liquid cultures were inoculated into 5 ml, triplicate samples of either TSB (bacteria) or Czapex Dox broth (yeast), supplemented with 0 (Control) or 100 µl (Treated) of aqueous extract from Tristar fenugreek leaves and examined at hourly intervals, all exhibited some stimulation of growth (Fig. 2). No evidence of growth inhibition was observed. This indicated that all accessions could be used in the development of cultivars for forage purposes. Bhatti et al. (1996) were able to demonstrate wide spectrum antibacterial activity against gram negative and gram positive bacteria, for both aqueous and ethanol extracts from fenugreek seed. Trigonella foenum-graecum L. seed purchased in Pakistan was extracted into either water or ethanol, and then used to make antibiotic discs, which prevented bacterial growth in zones surrounding each disc. Zones cleared of bacterial growth ranged in size from 12 to 21 mm, and exhibited a direct dose response relationship when different concentrations of the extracts were used. However, ethanol extracts from fenugreek seed purchased in India by De et al. (1999) did not inhibit growth of either bacteria or yeast. In our experiments, we examined aqueous and ethanol extracts from 75 different accessions of Trigonella species obtained from India, northern Africa, Europe and North America (Table 1) to determine if any of these lines possessed antibacterial or antifungal properties. Although antimicrobial activity has clearly been demonstrated for some fenugreek seed (Bhatti et al. 1996), none of the plants that we examined possessed this activity. However, Taylor et al. (2002) have shown that chemical composition can vary among different accessions of fenugreek originating from different countries of the world, as well as for plant lines grown at three different sites in western Canada over a 2-yr period. Variation in genetics and environmental response of plants to production of phytochemicals also has been shown for the Australian sweet lupin (Lupinus angustifolius), the Nigerian cowpea [Vigna unguiculata (L.) Walp], the Hawaiian kava (Piper methysticum Forster), and Capsicum annum L. (Ruiz et al. 1995; Oluwatosin 1999; Lebot et al. 1999; Zewdie and Bosland 2000). Processed food and nutraceutical industries which use plant additives such as fenugreek to enhance product value are responsible for providing labeling on their produce which reflects constituent contents and, addresses claims associated with active compounds found in their products. It is important to the economic development of these industries that genetic and ecotype variations in plants used by these industries are properly characterized, and that new plant lines with associated agronomic management packages capable of consistent production of important constituents are developed. Industrial products marketed as antimicrobials must be developed from plants showing genetic potential for antimicrobial activity. Our study indicates that under typical growth conditions used in western Canada, the 75 plant accessions examined lack any apparent antimicrobial activity, but are good candidates for further forage development. This study represents the most extensive survey of antimicrobial activity in Trigonella done to date. 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