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Published online 1 August 2005
Published in Crop Sci 45:1736-1740 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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FORAGE & GRAZING LANDS

Genotype and Environment Affect Rhizome Growth of Birdsfoot Trefoil

P. R. Beuselincka,*, E. C. Brummerb, D. K. Viandsc, K. H. Asayd, R. R. Smithe, J. J. Steinerf and D. K. Brauerg

a USDA-ARS, Plant Genetics Res. Unit, Univ. of Missouri, Columbia, MO 65211
b Agronomy Dep., Iowa State Univ., Ames, IA 50011
c Dep. Plant Breeding, Cornell Univ., Ithaca, NY 14853
d USDA-ARS, Forage and Range Res. Lab., Logan, UT 84322
e USDA-ARS, U.S. Dairy Forage Res. Ctr., Madison, WI 53706
f USDA-ARS, National Forage Seed Production Res. Ctr., Corvallis, OR 97331
g USDA-ARS, Dale Bumpers Small Farms Res. Ctr., Booneville, AR 72927

* Corresponding author (BeuselinckP{at}missouri.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Rhizome production has been transferred from wild germplasm of Lotus corniculatus L. (broadleafed birdsfoot trefoil) into domesticated germplasm to produce ‘ARS-2620’ and ‘ARS-2424’ (a L. corniculatus x L. uliginosus hybrid). The objective of this study was to determine if field environments in the United States differing in latitude affect rhizome expression in genotypes of ARS-2620 and ARS-2424. Ramets of rhizomatous genotypes of ARS-2620 and ARS-2424, and nonrhizomatous ‘Norcen’ were planted at seven locations in late July and August 1999. At five locations in 2000 and 2001, dormant plants were hand-dug in late autumn or winter. Traits measured were mean crown-plus-root mass, crown circumference, and percentage survival for all entries, and rhizome mass for the two rhizomatous entries. Significant (P ≤ 0.001) location x genotype effects were observed for all traits in 2000, but only for percentage survival in 2001. In 2001, the effect of locations was significant for all traits (P ≤ 0.001), but genotype effects were significant (P ≤ 0.001) only for crown circumference and percentage survival. The rhizomatous entries were larger in circumference and had a greater crown-plus-root mass than Norcen. Rhizomes appeared to be beneficial to plant survival and plant growth, but rhizomes did not assure performance or survival, as we did not identify a genotype that performed well across locations. High plant mortality and extensive tissue necrosis caused by root and crown-rot complex reduced overall plant performance and rhizome expression and masked the interpretive value of the data from the five locations. Plants grown in Logan, UT, were notable for their large size and rhizome production relative to the other locations, and as having advantageous, but undefined, environmental conditions beneficial to the performance of rhizomatous birdsfoot trefoil.

Abbreviations: masl, meters above sea level


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
LOTUS CORNICULATUS L. is a cross-pollinated, perennial legume used for pasture, hay, and silage production. Stand persistence of L. corniculatus depends on natural reseeding and individual plant persistence (Beuselinck, 1994). The value of rhizomes as a means to improve the persistence of birdsfoot trefoil had been conjectural until wild tetraploid (2n = 4x = 24) L. corniculatus germplasm expressing rhizomatous growth was discovered in the Atlas Range in Morocco (Beuselinck, 1989).

Beuselinck et al. (1996) reported that F1 hybrids derived from crossing Moroccan accessions with domesticated cultivars produced rhizomes in a field environment, but a sample of the same hybrids exhibited variable expression in the greenhouse (Beuselinck, 1994). Kallenbach et al. (2001) observed equal rhizome expression on plants grown from seed or ramets derived by clonal propagation. Variable expression of rhizomes between greenhouse and field environments was confirmed by Nualsri et al. (1998), who found incomplete rhizome (83%) expression in the greenhouse at 40 wk after planting, but complete expression in the field. Early reports from a performance trial conducted in Minnesota indicated that the rhizomatous cultivar ARS-2620 (PI 592503, Beuselinck and Steiner, 1996) failed to produce rhizomes (N.J. Ehlke, 1997, personal communication). Rhizome expression in birdsfoot trefoil has been shown to respond to short days (Nualsri et al.,1998; Kallenbach et al., 2001) and locations in the USA, with mild autumn and winter conditions providing more growing days that might be conducive to rhizome development.

Efforts to introgress the rhizomatous trait into new germplasm combinations have continued. Mating the nonrhizomatous L. corniculatus x L. uliginosus hybrid G4712 (2n = 4x = 24, AgResearch, Palmerston North, New Zealand) with five germplasm accessions (PIs 631539–542 and G31217) from Morocco generated a rhizomatous population that resulted in the germplasm ARS-2424 (PI 633724, Beuselinck, 2004) following selection and evaluation. ARS-2424 expands the germplasm resource for developing rhizomatous populations with diverse attributes, but the expression of rhizomes has not been determined outside the location where this germplasm was developed. The objective of this study was to determine if field environments in the United States differing in latitude affect rhizome expression in genotypes of ARS-2620 and ARS-2424.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Plant Materials
Ramets were vegetatively propagated by stem cuttings from 10 rhizomatous genotypes each of ARS-2620 and ARS-2424, and two genotypes of the L. corniculatus cultivar Norcen (Miller et al., 1983). Norcen does not produce rhizomes. The genotypes of the rhizomatous entries were chosen at random from among 40 genotypes of each entry previously verified to produce rhizomes in greenhouse and field environments at Columbia, MO. Single-stem rooted ramets were planted in the field at seven locations (Table 1) in late July and August 1999 and irrigated as needed for plant establishment. No fertility amendments were made before planting or during the study. All weak or dead ramets were replaced in late August 1999. The field design at each location was a completely randomized design with five ramets of each genotype randomized within each of two adjacent plantings. Each ramet of each genotype constituted a replication in each planting. Spacing was 1 m between rows and 0.5 m within rows. At each location in 2000, dormant plants in one planting were harvested in late autumn or winter after frost killed the herbage. The remaining planting was harvested in late autumn or winter 2001 after frost. Harvest dates varied by location as determined by timing of freezing conditions that killed the herbage and obvious plant dormancy. Weeds were mechanically controlled with the exception of the Arkansas location.


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  		Table 1. Site characteristics of five locations where rhizome expression of Lotus corniculatus entries was studied.

 
All surviving plants within the planting chosen for harvest in 2000 or 2001 were dug and soil and debris were removed from the roots and crown. Herbage was removed and discarded. Plants were express-shipped to Columbia, MO, for processing, where each plant was separated into crown, root, and rhizome portions. All stems were removed near their origin on the crown and discarded. Crown diameter was measured at top of the crown. Roots were trimmed to 12.7 cm below the top of the crown and discarded. The remaining root tissue was severed 5 cm below the top of the crown and constituted the root sample. Rhizomes were distinguished from roots as described by Li and Beuselinck (1996) and detached at their point of initiation from the crown. Distinguishing between rhizomes with chlorophyllic apices and stems derived from the crown was difficult, so rhizome samples were comprised only of rhizomes devoid of chlorophyll. Remaining vegetative tissues constituted crown samples. Crowns, roots, and rhizomes were oven-dried at 65°C for 10 d and weighed. Crown and root weights were combined for analysis. Percentage survival for each genotype was determined at time of harvest in 2000 and 2001 as the proportion of surviving ramets x 100. Percentage rhizome weight was calculated for each rhizomatous plant as a proportion of crown-plus-root weight x 100.

Data were analyzed for five of the seven locations as the integrity of the studies was jeopardized by long-tailed voles (Microtus longicaudus) in Oregon and by near complete mortality due to unmanaged weed competition in Arkansas. The data were analyzed using PROC GLM (SAS Institute, 1985) as a split-split plot with locations as main plots, years as subplots and genotypes as sub-subplots. The experimental unit was a single ramet of each of the 22 genotypes for a potential of n = 110 observations per year per location, n = 220 per location, and n = 1100 total for all five locations. Analyses of variance were performed on crown-plus-root mass, crown circumference, rhizome mass, percentage survival, and all possible interactions with main and subplots. Rhizome mass was analyzed only for the two rhizomatous entries. When significant F tests existed, mean separations were conducted for were each entry between locations within years using a protected LSD (P ≤ 0.05). Correlations between crown-plus-root mass, crown circumference, and rhizome mass were calculated using the correlation option within a combined ANOVA for the rhizomatous entries (SAS Institute, 1985). Multiple regression analysis was performed for site characteristics across locations (latitude, elevation, mean annual temperature, number of days with temperature ≥ 0°C, and annual precipitation) as independent variables on crown-plus-root mass, crown circumference, and rhizome mass combined during 2000 and 2001. Unless otherwise noted, all results are significant at the ≤0.05 probability level.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Location x genotype x year effects were observed for all traits in 2000, but only for percentage survival in 2001. In 2001, the effect of locations was significant for all traits, but genotype effects were present only for crown circumference and percentage survival. No genotype was identified that performed well across all locations.

There was a significant location x year effect on survival (Table 2). Percentage survival decreased between 2000 and 2001. Mortality was generally greater in Norcen than in ARS-2620 or ARS-2424. Survival of individual genotypes was not consistent among locations (data not shown). Year effects were confounded with plantings because plant survival measured in 2001 was based on plants that had not been measured in 2000. Genotype survival was not consistent between years. For example, Norcen survival in the New York location was 10% in 2000, but 30% in 2001.


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  		Table 2. Mean survival percentage of three Lotus cultivars at five locations in late autumn 2000 and 2001. Plants were established from rooted cuttings in late summer 1999.{dagger}

 
The crown-plus-root mass of all entries generally increased from 2000 to 2001 (Table 3). The mean crown-plus-root mass of ARS-2620 and ARS-2424 plants from Utah in 2001 significantly exceeded the weights of plants from the other four locations by >400 g. Considerable necrotic crown and root tissue was observed in plants grown at all locations. A greater level of tissue necrosis was observed in 2001 than 2000. Utah-grown plants were observed to be the least necrotic, while Missouri-grown plants were observed to be the most necrotic. Reduced crown-plus-root mass was indicative of smaller plants, but may also reflect a greater tissue necrosis.


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  		Table 3. Mean crown-plus-root mass of three Lotus cultivars at five locations in late autumn 2000 and 2001. Plants were established from rooted cuttings in late summer 1999.

 
Crown circumference of the entries generally increased from 2000 and 2001 (Table 4). In locations where all entries survived until the 2001 harvest, crown circumference was not different between the rhizomatous entries vs. the nonrhizomatous Norcen. Conspicuous among locations in 2001 was Utah, where ARS-2620 and ARS-2424 plants were {approx}3x greater in circumference when compared with plants in 2000. At the other locations, excluding Iowa, crown circumferences of plants in 2001 were approximately twice the size of plants in 2000. Plant circumference was positively related with root plus crown mass (r = 0.74, P ≤ 0.001, n = 589) when combined across all locations and entries.


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  		Table 4. Mean crown circumference of three Lotus cultivars at five locations in late autumn 2000 and 2001. Plants were established from rooted cuttings in late summer 1999.

 
A significant location x year effect was observed for yield of rhizome mass. Rhizome mass was significantly greater at Utah than the other locations in 2000 and 2001 (Table 5). Rhizomes were produced by ARS-2620 and ARS-2424 at all locations and both years, but not observed on any Norcen plants. Rhizome mass as a proportion of crown-plus-root mass was similar both years for ARS-2620 and ARS-2424 by location (Table 6). ARS-2424 produced a greater proportion of rhizome mass relative to crown-plus-root mass at Utah in both years. Rhizome mass was positively related with crown-plus-root mass (r = 0.42, P ≤ 0.001, n = 571) and plant circumference (r = 0.50, P ≤ 0.001, n = 571) when combined across all locations and the two rhizomatous entries.


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  		Table 5. Mean dry mass of rhizomes of two rhizomatous Lotus cultivars at five locations in late autumn 2000 and 2001. Plants were established from rooted cuttings in late summer 1999.

 

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  		Table 6. Mean percentage of rhizome dry mass of crown-plus-root dry mass of two rhizomatous Lotus cultivars at five locations in late autumn 2000 and 2001. Plants were established from rooted cuttings in late summer 1999.

 
Location elevation (m) was the only site characteristic variable that provided significant linear equations for crown-plus-root mass (y = –35.4 – 0.20x; R2 = 0.20), crown circumference (y = –82.9 + 0.029x; R2 = 0.23), and rhizome mass (y = –14.1– 0.019x; R2 = 0.31) using only data for ARS-2620 and ARS-2424 combined over all locations in 2000 and 2001. Residual analysis of the data demonstrated that data from the Utah location skewed the data to generate significant, but trivial, predictive equations. Removing the Utah location from the regression analysis for rhizome mass retained location elevation as the only site characteristic variable to fit a significant linear equation (y = –11.3 – 0.023x; R2 = 0.12). Number of days with temperature ≥ 0°C at the locations, excluding the Utah location, was the only site characteristic variable that provided significant linear equations for crown-plus-root mass (y = –298.4 + 0.76x; R2 = 0.07) and crown circumference (y = –89.4 + 0.162x; R2 = 0.02). Low R2 values for the predictive equations did not provide any insight into the effect of site characteristics on the performance of ARS-2620 and ARS-2424.


   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Genotype x location effects are common in birdsfoot trefoil (McGraw et al., 1986; McGraw and Beuselinck, 1990). Performance of genotypes at any given location are not indicative of performance elsewhere. Using vegetative propagations of known rhizome-producing genotypes allowed us to replicate individuals to study their expression of rhizomes across many locations. Using genetically identical ramets of genotypes permits the isolation of any environmental effects on rhizome expression from the genotype's genetic constitution. This type of study would not be possible using only seed-derived plants. Within a seed-derived population of birdsfoot trefoil plants expressing rhizomes, trait expression would be subject to the unique genetic constitution of individual genotypes generated through cross-pollination interacting with the environment, but no replication would be possible.

Mortality of birdsfoot trefoil plants has been associated with genetic differences among genotypes and their resistance to disease and/or environmental stresses (English, 1999). Plants severely infected with crown and root rot exhibit extensive tissue necrosis in the central portion of the upper taproot and crown. Mortality of 68 to 88% of stands by the end of the second year were reported by Henson (1962), who found >80% of surviving plants badly diseased by crown and root rot; and 90% of stands were lost in 2 yr in Missouri regardless of management (Beuselinck et al., 1983). Root and crown morphologies of field-grown plants established from vegetative propagules or seed are similar, as crown- and root-rot complex causes altered root morphology and extensive tissue necrosis. Previous studies describing birdsfoot trefoil mortality used nonrhizomatous populations of birdsfoot trefoil, because no rhizome-bearing populations have been available until recently (Emery et al., 1999).

Rhizomes can affect plant perennation through vegetative reproduction in many species (Li and Beuselinck, 1996). Rhizomes serve as storage organs that are protected below the soil surface and contribute to higher stored carbohydrate availability during winter dormancy and the spring regrowth periods (Nualsri et al., 1998). Only 10% of the Norcen plants survived into the second year of our study, compared with 40% of ARS-2620 and ARS-2424. The greater survival of ARS-2620 and ARS-2424 relative to Norcen likely reflects enhanced survival resulting from rhizomes, which are absent in Norcen. Although rhizomes appeared to be beneficial to plant survival and plant growth, rhizome expression was variable in the different locations of this study. Rhizome mass accounted for little of the dry mass of rhizomatous entries. In late autumn, distinguishing between rhizomes with chlorophyllic apices and stems derived from the crown can be difficult. Our collection of only those rhizomes devoid of chlorophyll might have resulted in a conservative estimate of rhizome expression.

Rhizomatous entries were larger in circumference and had a greater crown-plus-root mass than Norcen. Rhizome mass is greater on larger plants as evidenced by the the positive correlations with crown-plus-root mass (r = 0.42) and plant circumference (r = 0.50). Because large birdsfoot trefoil plants are generally considered to be healthier and less subject to stress-induced mortality (Drake, 1961; Emery et al., 2003), rhizomes might be advantageous to longevity, even if only for one additional season. Minor increases in plant longevity can contribute to improved stand persistence in this reseeding legume. Additionally, extensive tissue necrosis was noted in rhizomatous and nonrhizomatous plants. Genotypes with enhanced disease resistance or winterhardiness may also be longer lived, regardless of rhizome expression. Thus, our findings cannot distinguish if greater plant survival was attributable to rhizomes.

Rhizome expression in birdsfoot trefoil has been shown to respond to short days (Nualsri and Beuselinck, 1998; Kallenbach et al., 2001). If rhizomes initiate during short days, it is logical that locations with more days of lighting and growing conditions conducive to rhizome expression would result in greater rhizome production. We hypothesized that rhizome expression would be sensitive to the latitude differences at the different locations used in this study. Although there was a significant location effect for rhizome mass, we did not discover evidence to support our latitude sensitivity hypothesis. The variable growth conditions of the locations of this study, like daylength, length of growing season, temperature, and moisture intuitively affected differences in rhizome production, but we were not able to identify a single site characteristic that could explain the differences among locations. High plant mortality and extensive tissue necrosis caused by root and crown-rot complex reduced overall plant performance and rhizome expression and masked the interpretive value of the data from the five locations. The original plans of our study included locations in Oregon and Arkansas that could have provided data important to understanding rhizome expression in birdsfoot trefoil.

The different growth of plants at Utah vs. the other four locations demonstrates the effect of environment on rhizome expression. This location is not much farther south than the Iowa location, but is {approx}1100 masl (meters above sea level) higher in elevation. The short growing season at the Utah location is typified by high solar radiation, arid days, and cool nights. These conditions are not dissimilar to sites where the rhizomatous L. corniculatus germplasm was discovered in Morocco at {approx}33° N lat. and elevations of 1490 to 1855 masl (Beuselinck, 1989). Our analyses were not successful identifying site characteristics that could explain the better performance of ARS-2620 and ARS-2424 at this location. We observed that Utah-grown ramets of genotypes had the least amount of necrotic crown and root tissues, suggesting that the combined cool and arid conditions may not favor the growth of root- and crown-rot disease complexes that weaken and kill birdsfoot trefoil (Drake, 1961; English, 1999).


   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
We used vegetative propagules of birdsfoot trefoil genotypes known to produce rhizomes to study the expression of rhizomes in the same set of genotypes across many locations. When using vegetative ramets, considerable environmental effects were noted on the expression of crown-plus-root mass, rhizome mass, crown circumference, and plant survival. Birdsfoot trefoil plants producing rhizomes may be larger, but rhizomes did not assure performance or survival, as we did not identify a genotype that performed well across locations. Although there was a significant location effect for rhizome mass, we did not discover evidence to support our latitude sensitivity hypothesis. High plant mortality and extensive tissue necrosis caused by root and crown-rot complex reduced overall plant performance and rhizome expression and masked the interpretive value of the data from the five locations. An analysis of site characteristics did not explain the performance differences between ARS-2620 and ARS-2424 among locations. However, the Utah location was identified as having advantageous, but undefined, environmental conditions beneficial to the performance of rhizomatous birdsfoot trefoil. Variable mortality among genotypes and ramets of genotypes in this study exposes the interaction between genotypes and environment that typically goes unnoticed in rhizomatous and nonrhizomatous populations of this naturally reseeding crop. Additional studies are needed to elucidate the value of rhizomes to plant longevity and performance of rhizomatous birdsfoot trefoil affected by the disease complex known as root and crown rot.


   ACKNOWLEDGMENTS
 
We thank Dr. Mark Ellersieck, statistician for the Missouri Agric. Exp. Stn., for his statistical advice and assistance with the analysis and interpretation of our data.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Contribution of the Missouri Agric. Exp. Stn. This research was a joint collaboration under the project NE-144, "Forage Crop Breeding to Improve Yield and Quality." Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the USDA or the Univ. of Missouri and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

Received for publication August 17, 2004.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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