168 D.W. Lucero et al. European Journal of Agronomy 11 1999 167–177
1. Introduction
Preference at the Ecole Nationale Superieure d’Agronomie
et des
Industries Alimentaires
White clover Trifolium repens L. is commonly ENSAIA, Nancy, France. In March of 1995
used as a perennial forage legume in the temperate seedlings were grown in specially constructed PVC
zones of both hemispheres. Usually, white clover is tubes and boxes described below. Three composi-
grown with one or more grass companions, the most tions of white clover and ryegrass plant interaction
common being perennial ryegrass Lolium perenne were combined with three levels of soil water
L. Hill and Michaelson-Yates, 1987. Grown deficit in a randomized complete block experiment
together, white clover and ryegrass are major contrib- with four replications. White clover was inoculated
utors to the productivity and quality of pastures. with Rhizobium triflii SB116 on day 10. From
In mixture, grasses and legumes compete for week four to nine a complete nutrient fertilizer
water, light and nutrients and may also compete for solution Robin et al., 1992 was applied once per
O 2
, CO 2
and space Haynes, 1980. Mixture studies week. This nutrient solution was slightly modified
have often looked at the effects of cutting and in order to supply both white clover and ryegrass
grazing Wilman and Asiegbu, 1982; Evans and with N, P, and K at rates equivalent to
Williams, 1987; Woledge et al., 1992a, inorganic N 165 kg ha−1, 60 kg ha−1 and 60 kg ha−1, respec-
and temperature Davidson and Robson, 1986; tively. Average daily maximum and minimum air
Ledgard, 1991; Nesheim and Boller, 1991 and light temperatures
were 26±4°C
and 13±3°C,
and photosynthesis Dennis and Woledge, 1985; respectively.
Thompson and Harper, 1988; Woledge et al., 1992b, as well as competition Martin and Field, 1984;
2.2. Plant interaction treatments Menchaca and Connolly, 1990; Lu¨scher et al., 1992.
The dynamics of water availability and use are White clover and ryegrass were grown as fol-
essential factors differentiating plant survival in a lows: 1 individually no interaction; 2 shoot
pasture. Little, however, is known concerning only interaction shoot interaction; and 3 both
white clover and ryegrass response to water deficit root and shoot interaction shoot+root inter-
in a mixed pasture. Reduced white clover growth action. White clover and ryegrass grown in no
in pasture mixtures under drought has been attrib- uted to its less extensive rooting system Thomas,
interaction were seeded into plastic PVC tubes 1984; Guobin and Kemp, 1992. Yet the capacity
5 cm in diameter and 30 cm in depth filled with of white clover to extract water from deep soil
soil made of a quartz sand filtered to pass a 2 cm reserves has also been reported Guckert et al.,
sieve added to a potting mix at a weight propor- 1993. Therefore, the results concerning white
tion of 70:30 and packed to simulate a bulk density clover are conflicting. This greenhouse study was
of 1.25±0.10 g cm−3. Individual PVC tubes were conducted to determine the effects of soil water
spaced at intervals of 20 cm. For shoot interaction, deficit and interspecific plant interaction on the
20 PVC tubes 10 white clover and 10 ryegrass dry matter DM yield of white clover and ryegrass
plants, identical in nature to those containing no and on the morphogenesis of white clover. White
interaction plants, were placed in a 5×4 grid in an clover and ryegrass were grown in a shallow soil,
alternative pattern in specially constructed crates either individually no interaction or in mixtures
30 cm×24 cm×30 cm. The 14 plants on the of equal numbers with just shoot interaction or
outer edge of the 5×4 grid were defined as border with both shoot+root interaction.
plants and were not included in experimental meas- urements. Each PVC tube was placed in a drilled
hole of diameter slightly larger than the PVC tube
2. Methods and materials
itself. Holes were drilled in a sheet of 5 mm ply- wood. The plywood sheet was supported to the
2.1. Experimental conditions same height as the lip of each PVC tube. In this
manner the 5 mm plywood sheet became the sup- A greenhouse experiment was conducted with
white clover cv. Huia and perennial ryegrass cv. port for above-ground canopy growth, and acted
169 D.W. Lucero et al. European Journal of Agronomy 11 1999 167–177
as the artificial soil level. Seeds of white clover and ber Scho¨lander et al., 1965 at three dates in order
to verify plant water stress levels. Soil water deficit ryegrass were sown in the center area of each tube,
hence the spacing between plants was 6 cm. Thus, levels were obtained at day 66 after sowing. Tubes
and boxes containing plants were thereafter while the above-ground canopy was allowed to
grow together, the PVC tubes kept the below- weighed every 2 days and at that time the water
lost to transpiration was added in order to bring ground plant parts completely separated. In the
shoot+root interaction treatment, white clover and tube and box weights back to soil water deficit
levels due to plant cover soil evaporation was ryegrass were seeded not into plastic PVC tubes
but into plastic boxes of the same dimension as negligible. For both no interaction and shoot inter-
action treatments, the tubes containing white clover the shoot interaction crates, i.e. 30 cm×24 cm×
30 cm in an identical number 10 white clover or ryegrass were weighed individually. For plants
grown in shoot interaction this required the careful and 10 ryegrass plants and in an identical pattern
to that of plants in the shoot interaction treatment lifting up by approximately 1 cm of the tube
containing the plant in such a manner as to 5×4 grid, 6 cm spacing. Hollow plastic PVC
tubes identical to those in which no interaction and minimize disturbance of the developed above-
ground canopy. Once weighed, the tube containing shoot interaction plants were grown were sealed at
both ends and placed alternatively between the the plant was gently re-lowered into the above-
ground undisturbed canopy. For the shoot+root seeded white clover and ryegrass plants in the
plastic boxes of the shoot+root interaction treat- interaction treatment, boxes were weighed in
entirety. Once soil water deficit levels were ment. These hollow PVC tubes were thus placed
in order to both simulate similar rooting conditions obtained, plants were subjected to soil water defi-
cits for a 37 day period before harvest. and to assure that the soil volume per plant was
identical for each of the three plant interaction treatments. Plants in the shoot+root interaction
2.4. Dry matter yield treatment were allowed to develop with both
above- and below-ground plant parts in complete Two replications 108 total plants were har-
vested on day 102 and the other two on day 103 interaction.
after sowing. All plants were separated into above- and below-ground material. Above-ground white
2.3. Soil water deficit levels clover plant parts were further separated into leaf
lamina, petioles, stolons and flowers. Roots of Soil water deficit levels were allowed to develop
gradually by the progressive withdrawal of water both white clover and ryegrass were carefully
washed with a jet of tap water to remove soil. In at day 50, at which time the leaf area index LAI
for the shoot interaction and the shoot+root inter- order to minimize root loss during washing the
plant roots were individually rinsed in wooden action treatments was approximately 3. Pre-experi-
mental tests using identical PVC plastic tubes, boxes with a base made of a 2 mm mesh screen
and the root material separated during the washing boxes and soil had determined the soil weight
70 and 55
of field capacity, respectively per process was carefully gathered and included for
plant root DM measurements. Harvested plant tube and box necessary to obtain the desired plant
water stress levels. Plant water stress levels were parts were immediately dipped in liquid nitrogen,
stored at −30°C until freeze drying and weighed. based on pre-dawn leaf water potentials of white
clover and ryegrass, i.e. no −0.0 MPa, moderate The harvest of the shoot interaction and the
shoot+root interaction treatments necessitated the −0.5 MPa and severe −1.0 MPa. Soil water
deficits corresponding to plant stress below careful separation of the above-ground canopy.
Measurements were carried out on the six plants −
1.0 MPa drastically reduce growth and can lead to plant death Guckert et al., 1993. During the
in the inner 12×18 cm 2 experimental area. For the
shoot+root interaction treatment, root parts also experiment, pre-dawn leaf water potentials were
measured just before sunrise with a pressure cham- needed to be separated. Overall, a total of 12 white
170 D.W. Lucero et al. European Journal of Agronomy 11 1999 167–177
clover and 12 ryegrass plants were harvested for each interaction treatment and soil water deficit
level.
2.5. Morphological measurements Non-destructive measurements [stolon length,
internode length, primary leaf appearance rate LAR and secondary LAR] were carried out on
two randomly selected stolons from two white clover plants per replication 72 plants in total,
eight plants per interaction and soil water deficit level . Measurements for time zero were taken at
the establishment of water stress day 66 and thereafter on days 77, 88, 96 and 102. Primary and
secondary LARs were determined according to criteria established by Carlson 1966, and total
leaf index was a combination of both primary and secondary LARs [see Fig. 2a–c]. Primary leaves
were defined as those leaves growing from the primary stolon axis and secondary leaves were
defined as those leaves developing on stolons initi- ated from the main axis of selected stolons. The
relative growth rate RGR of stolons and leaves was a function of growth divided by time
[Figs. 1c, 2a–c]. At each measurement, stolon length, from one internode to another, was
recorded and average internodal length was a
Fig. 1. Stolon length, stolon internodal length and stolon rela-
function of the lengths of all internodes summed
tive growth rate RGR of white clover plants grown individu-
and divided by the number of internodes.
ally, in shoot+root interaction, or just shoot interaction with ryegrass and subjected to three levels of water deficit. Error
bars on observed data indicate ±1 standard deviation.
2.6. Statistical analysis The effect of treatments was obtained from
highest root DM yield, and plants grown in shoot+root interaction the lowest root DM yield.
analyses of variance. Mean separations were per- formed by an LSD procedure where the F values
For ryegrass grown in no interaction, an increase in soil water deficit from no to moderate reduced
were significant at the 0.05 probability level SAS Institute, 1990.
root DM yield by over 50 and an additional
increase to severe soil water deficit further reduced root DM yield by 36
.
3. Results
