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a Dep. of Agron., Univ. of Missouri, Columbia, MO 65211
b USDA-ARS, Plant Genet. Res. Unit, Columbia, MO 65211
* Corresponding author (BeuselinckP{at}missouri.edu)
Received for publication October 5, 2001.
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONREFERENCES |
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Abbreviations: CE, catechin equivalent • CT, condensed tannin • DM, dry matter • NIRS, near infrared reflectance spectroscopy
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONREFERENCES |
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McLeod (1974) reported a comprehensive review of the effects of tannins from various plants on ruminants. High concentrations of CTs depress voluntary feed intake, digestive efficiency, and animal productivity (Barry and Duncan, 1984; Aerts et al., 1999). High CT concentration is also associated with high lignin, low crude protein, and low in vitro digestible dry matter (DM) concentration (Miller and Ehlke, 1994). On the other hand, low to moderate concentration of CTs have beneficial effects on livestock. Condensed tannins reduce bloat and increase protein absorption in grazing ruminants (McLeod, 1974). Protein in forage legumes is often poorly utilized by ruminants because of extensive degradation in the rumen. Condensed tannins facilitate the bypass of protein that might otherwise be lost through microbial deamination in the rumen (Barry et al., 1986; Tanner et al., 1994). This bypass is made possible by reactive components of CTs, which complex with soluble proteins, making them insoluble at rumen pH (pH 5.8–6.8) but soluble and released at the more extreme pH conditions found in the abomasum (pH 2.5–3.5) and small intestine (pH 7.5–8.5) (Barry and Manley, 1984). This process increases the absorption of essential amino acids in the small intestine (Waghorn et al., 1987). It has been shown that moderate CT concentrations increased lactation, wool growth, and live weight gain, without reducing voluntary feed intake (Aerts et al., 1999). Condensed tannins can also improve animal health by overcoming effects of gastrointestinal parasites (Aerts et al., 1999). The desirable concentration of CTs for ruminants should represent a balance between the positive and negative effects of tannins. An optimum concentration has been suggested to be 20 to 40 g kg-1 forage dry weight (Barry et al., 1986; Aerts et al., 1999).
Perennial Lotus spp.—birdsfoot trefoil, narrowleaf trefoil, and big trefoil—are used in major forage production centers of the world (Papadopoulos and Kelman, 1999). The three species are markedly different in their genetics, morphology, and environmental adaptation. Birdsfoot trefoil is the most important Lotus sp. in North America, covering an area greater than 1 million ha (Beuselinck and Grant, 1995). Birdsfoot trefoil is a tetraploid (2n = 4x = 24) distributed throughout the world with a wide range of environmental adaptation. Big trefoil is a rhizomatous diploid (2n = 2x = 12) with a narrow geographic distribution; it's particularly well adapted to low, wet, and waterlogged habitats (Papadopulos and Kelman, 1999). An autotetraploid form (2n = 24) is available as a commercial cultivar. Narrowleaf trefoil is also a diploid (2n = 2x = 12) with a woody tap root and tolerates infertile, acidic, and poorly drained soils (Beuselinck and Grant, 1995).
Even though birdsfoot trefoil, narrowleaf trefoil, and big trefoil have great potential as pasture species from an agronomic point of view, realizing that potential in the grazing animal will depend partly on CT concentration. Previous work indicated that CT concentration varies among species and cultivars (Kelman and Tanner, 1990; Roberts et al., 1993), and CT may also differ in structure and biological activity (Foo et al., 1996). Concentration of CTs is also influenced by environmental factors (Barry and Forss, 1983). Seasonal CT changes and its partitioning in different plant parts of the three widely grown Lotus spp. has not been investigated. Newly developed cultivars and germplasms of Lotus spp. destined for use by producers need to be evaluated for CT concentration. Knowledge of CT concentration and an understanding of the morphological and seasonal CT variation of Norcen, ARS-2620, ARS-1221, and ARS-1207 are important to modify feeding practices or initiate genetic improvement projects. Also, the availability of rapid techniques for ranking species or genotypes of Lotus according to CT concentration is useful to identify potential candidates for inclusion in genetic improvement projects. The objectives of this research were (i) to determine the level of CT concentration and its seasonal variations in three widely grown Lotus spp.; (ii) to assess the distribution of CTs in leaves, stems and flowers, and (iii) to determine if herbage of greenhouse-grown Lotus spp. contains CT concentrations with equivalent rank to field-grown plants.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONREFERENCES |
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Plantings I and II were arranged in a randomized complete block design with three replications where species were main-plot treatments. Plants were spaced 1 m between rows and 0.5 m within a row. Annual and perennial weeds were controlled by applying hexazinone [3-cyclohexyl-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4 (1H,3H)-dione] in midwinter when plants were dormant. Further weed control was performed by spot application of glyphosate [N-(phosphonomethyl) glycine] and hand weeding.
Sampling Procedure
Established plants were sampled in the following spring, summer, and fall after planting. The sampling dates of Planting I were 10 June, 25 Aug., and 21 Oct. 1997; Planting II was sampled on 10 June, 2 Sept., and 14 Oct. 1998. Five plants from each treatment and each replication were randomly chosen and dug, and shoots were mechanically separated from crown. The five plants were mixed to form one sample for CT analysis.
In late spring of 1997 and 1998, an additional five plants of ARS-2620, ARS-1207, and ARS-1221 were harvested and hand-separated into leaves, flowers (umbels), and stems. Norcen was not included at the time of sampling because it had not flowered. All samples were packed in ice and transported to the laboratory. Samples were kept frozen at -4°C until freeze-dried.
Greenhouse Study
A greenhouse study was initiated in 1998 and repeated in 1999 to determine if differences in CT concentration observed in field planting were similar to greenhouse-grown plants. Scarified seeds of Norcen, ARS-2620, ARS-1207, and ARS-1221 were planted in mid-April in 1-L plastic pots filled with commercial growth medium as previously described. Each entry was replicated eight times and arranged in a completely randomized design. Seedlings were thinned to one plant per pot and allowed to grow 12 wk under natural lighting in the greenhouse. Pots were watered as needed. Insect pests were controlled in the greenhouse with imidacloprid [1-(6 chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-yilidiniamine]. In mid-August, herbage was harvested to 5 cm from soil and stored at -4°C until freeze-dried.
All field and greenhouse samples were freeze-dried and then ground in a Udy cyclone mill (Model 3010-018, Udy Corp., Fort Collins, CO) to pass a 1-mm screen. Ground samples were stored in sealed containers at -20°C until analyzed for CTs.
Near Infrared Spectroscopy Analysis of Condensed Tannins
Condensed tannin concentration was determined by NIRS as described by Roberts et al. (1993). All samples were scanned from 1100 to 2500 nm with a scanning monochromator (NIRSystems 5000, Foss-NIRSystems, Silver Spring, MD), and spectra were recorded and modified with software developed by Infrasoft International (Port Matilda, PA). A subset of 90 samples was selected on the basis of spectral diversity, and those samples were analyzed chemically for total CTs as described below. Chemical data were used to develop a prediction equation using modified partial least squares regression. The optimum NIRS equation was selected on the basis of squared correlation coefficients of calibration (R2), 1 - the variance ratio, and low standard errors of calibration and cross validation (Table 1).
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Statistical Analyses
The error variances of 1997 and 1998 CT data of field experiments and 1998 and 1999 CT data of greenhouse experiments were determined to be homogeneous according to Bartlett's test (Steel and Torrie, 1980). Thus, the 2 yr of CT data of respective experiments were combined in the ANOVA. The field study was analyzed as a split-split plot where year was main plot, species was subplot, and season or plant parts were sub-subplot. The greenhouse experiment was analyzed as a split plot where year was main plot and species subplot. The data were analyzed using the general linear model (GLM) of SAS version 6.0.3 (SAS Inst., 1988). Means were separated using Fisher's protected LSD and considered to be significant at P = 0.05.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONREFERENCES |
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Field Study
All entries established and grew well in both testing years as evidenced by large crowns, extensive vegetative growth, and well-developed root systems. Mean CT values differed among entries and seasons, and there was a significant entry x season interaction both years. Field herbage samples of big trefoil ARS-1221 germplasm had the greatest concentration of CTs, averaging 154 g CE kg-1 DM (Fig. 1)
. Herbage of narrowleaf trefoil ARS-1207 had the lowest CT concentration, averaging only 8 g CE kg-1 DM. Birdsfoot trefoil cultivars Norcen and ARS-2620 had moderate CT levels.
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The CT concentrations of Norcen and ARS-2620 (Fig. 1 and 2) were within the range reported for birdsfoot trefoil by other researchers (Roberts et al., 1993; Miller and Ehlke, 1994). Even though both ARS-2620 and Norcen belong to the same species, ARS-2620 had 60 to 70% more CTs than Norcen. The two entries are also morphologically distinct in that ARS-2620 is rhizome forming and flowers 3 to 4 wk earlier than Norcen.
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Soil nutrient status is one of the environmental factors that influences CT content of Lotus spp. Barry and Forss (1983) reported that big trefoil cultivar Grasslands Maku had CT concentration of 80 to 110 g kg-1 DM when grown in acid soils without fertilizer and only 20 to 30 g kg-1 DM when grown in high-fertility soil. The differences in CT concentration among Lotus spp. in this study were not induced by low soil fertility. In our study, the plants were grown in a fertile silt loam soil; thus, soil nutrients were not limiting and unlikely to be a major factor influencing CT concentrations.
Seasonal Changes
There was a significant seasonal variation in CT concentration of all entries except ARS-1207. Mean herbage CT concentration of ARS-1207 was similar among spring, summer, and fall samples with a slight peak in summer (Fig. 1). Inherently low-tannin plants show little seasonal variation of CT concentration. Roberts et al. (1993) reported that low tannin accessions of birdsfoot trefoil tested at two locations showed little or no change in CT concentration in July, August, and September. In sericea lespedeza [Lespedeza cuneata (Dum.-Cours.) G. Don.] greater seasonal CT fluctuations were reported for high- than low-tannin entries (Fales, 1984; Windham et al., 1988).
Norcen, ARS-2620, and ARS-1221 had greater herbage CT concentration in spring and summer than fall harvest. Generally, mean spring and summer herbage CT concentration of these entries were twice the level observed in fall (Fig. 1). The seasonal changes in CT concentration for these entries generally agree with reports by Roberts et al. (1993), who demonstrated that CT concentration declined by 40% from July to September in high-tannin accessions of birdsfoot trefoil. Similarly, in high-tannin sericea lespedeza entries, there were marked differences in CT concentration between summer and fall (Windham et al., 1988).
The relatively high temperatures of spring and summer are thought to induce the formation of additional CTs. Lees et al. (1994) reported that big trefoil clones grown under high temperature (30°C) had substantially greater levels of tannin than the same clones grown under cooler temperature (20°C). Sericea lespedeza cultivars grown under a warm temperature regime (32 and 24°C, day and night) had CT concentrations 100 g kg-1 DM greater than those grown at a cool temperature (22 and 17°C, day and night) (Fales, 1984). In addition to its high-tannin content, the quality of spring- and summer-harvested herbage is expected to be low compared with fall. Environmental stresses that stimulate the shikimic acid pathway provide precursors for both CT and lignin in plants (Barry and Manley, 1986).
We did not expect CT concentration of ARS-1221 to be less in summer than in spring although it was well above 150 g CE kg-1 DM in summer. The lower herbage CT concentration of ARS-1221 in summer was probably due to prolonged heat stress, which induced the breakdown of previously formed CT. Senescing and chlorotic lower leaves appeared more visible in ARS-1221 than in the other entries. Lees et al. (1994) reported that CT vacuoles were rare in subepidermal layers of chlorotic leaves of big trefoil, indicating the curtailment of tannin production. Cooler temperatures in the fall were also less favorable for the production of tannins, resulting in a 40 to 50% decline of CT concentration for Norcen, ARS-2620, and ARS-1221 herbage. In addition to cooler temperature effects, the decline of CT concentration in the fall, compared with spring and summer, could be attributed to extensive cross-linking of CT polymer proanthocyanidins and leucoanthocyanidins (Briggs, 1990).
The variation in CT concentration in Norcen, ARS-2620, and ARS-1221 in spring and summer vs. fall has important implications with respect to animal performance. Barry et al. (1986) recommended that a CT concentration of 20 to 40 g kg-1 DM in Lotus spp. would represent a balance between the positive effect of improving N digestion and the negative effect of depressed intake and carbohydrate digestion in the rumen. On the other hand, Miller and Ehlke (1994) showed that CT concentrations in birdsfoot trefoil as high as 85 g CE kg-1 DM produced little or no reduction in DM digestibility. In our study, regardless of season, tannin levels of Norcen and ARS-2620 were within or close to the ranges suggested by Barry et al. (1986) and Miller and Ehlke (1994).
In contrast, CT concentration of ARS-1221 was high enough, even in the fall, to reduce herbage intake and digestibility. Barry and Duncan (1984) reported that CT concentration in big trefoil more than 100 g kg-1 DM depressed metabolizable energy intake of sheep (Ovis aries) due to depression of both voluntary intake and digestion of organic matter. In our study, fall-harvested herbage of ARS-1221 had a CT concentration of more than 100 g kg-1 DM. Herbage CT concentration of ARS-1207 was consistently below either range suggested above and may need to be elevated to maximize animal performance.
This study indicates that, if ARS-1221 is to comprise a major portion of animal feed, interference either through management or breeding is necessary to alter the CT concentration to a desirable level. It has been shown that CT concentration of Lotus spp. can be altered through breeding and selection. Miller and Ehlke (1997) determined that CT concentration was controlled mainly by additive genetic effects in tannin-positive populations, and mass selection would be effective in increasing or decreasing herbage CT concentration. Unlike birdsfoot trefoil, which is a tetraploid, both ARS-1221 and ARS-1207 are diploids. Thus, it should be easier to genetically manipulate tannin levels in ARS-1221 and ARS-1207 relatively to birdsfoot trefoil. Because both ARS-1221 and ARS-1207 are random-mated heterogenous populations, there should be sufficient genetic variation to identify and select plants with desirable tannin levels.
Plant Components
Concentration of CTs varied significantly among plant components of ARS-2620, ARS-1207, and ARS-1221 (Fig. 2). The pattern of CT distribution was also different in each species. Generally, flowers had the greatest concentration of CT, except in ARS-1221 where CT levels of leaves and flowers were the same. Both leaves and flowers of ARS-1221 had CT concentrations greater than 200 g kg-1 DM, which has not been reported previously for field-grown big trefoil. Lees et al. (1994) reported leaf CT concentrations ranging between 112 and 227 g kg-1 DM for big trefoil, but this was in a controlled environment.
Stem tissues, in general, had the least concentration of CTs. Stems of ARS-1207 were tannin free while those of ARS-2620 had less than 10 g CE kg-1 DM. However, unlike ARS-2620 and ARS-1207, stems of ARS-1221 were rich in CTs, averaging 130 g CE kg-1 DM. The CT distribution of ARS-1221 also differed from ARS-2620 and ARS-1207 where higher CT concentrations were found in flower tissue than in leaves or stems. It has been suggested that high-CT concentration in the flowers is a defense strategy for the reproductive tissues (Terrill et al., 1992). We have identified in other research that a greater concentration of hydrogen cyanide, another defense molecule, is found in birdsfoot trefoil flowers (Gebrehiwot and Beuselinck, 2001).
Greenhouse Study
Differences in CT concentrations among the four entries grown in the greenhouse were significant both years (Fig. 3)
. As in the field study, the two diploid species, ARS-1221 and ARS-1207, had the highest and lowest CT concentrations, respectively. Averaged over 2 yr, herbage of ARS-1221 had CT concentration of 113 g CE kg-1 DM while that of ARS-1207 was only 8.5 g CE kg-1 DM (Fig. 3). Tannin levels of Norcen and ARS-2620 were lower than those of ARS-1221 but higher than those of ARS-1207. We also found a significant difference in CT concentration between ARS-2620 and Norcen. Herbage of ARS-2620 had three times as much CT as Norcen. Mean CT concentrations were 60 and 20 g CE kg-1 DM for ARS-2620 and Norcen, respectively.
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SUMMARY AND CONCLUSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONREFERENCES |
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Seasonal CT differences were significant for the entries Norcen, ARS-2620, and ARS-1221. More CTs were found in spring and summer herbage of Norcen, ARS-2620, and ARS-1221 than in fall. The level of CTs in ARS-1207 did not show much seasonal fluctuation and remained below the desired CT concentration. On the other hand, CT concentration of ARS-1221 was high at all sampling times. Thus, a lower CT concentration of ARS-1221 would minimize the negative consequences of high CT and may be achieved through breeding or feed management such as supplementation with tannin-binding polymers.
In all species, leaves and flowers had more CTs than stems. However, the pattern of CT distribution varied among species. Flowers, leaves, and stems of ARS-1221 were very high in tannin. In contrast, flowers of ARS-2620 and ARS-1207 had greater CT concentrations than did leaves or stems. Both ARS-1207 and ARS-1221 appear to be suitable candidates for genetic improvement to respectively increase and decrease CT concentrations to a desirable level. Because CTs are unevenly distributed in plant organs, breeding efforts to alter CT concentration would require close attention to plant parts.
Rankings of the birdsfoot trefoil, narrowleaf trefoil, and big trefoil entries for CT concentration were the same in the field and greenhouse. Except for ARS-1221, actual CT values were also similar between field- and greenhouse-grown plants. Thus, these Lotus spp. can be screened in the greenhouse for CT concentration for breeding purposes. Achieving equivalent tannin expressions in big trefoil in both the field and greenhouse may require additional manipulation of the environmental parameters for greenhouse-cultured plants.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONREFERENCES |
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONREFERENCES |
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