chung1995

Published July, 1995
Effect of Alfalfa Plant and Soil Extracts on Germination and Growth of Alfalfa
111-Min Chung and Darrell A. Miller*
ABSTRACT
cubated at room temperaturefor 1 wk. Residues from several alfalfa cultivars were equally toxic to alfalfa seed germination, regardless of saponin content. This led to the
conclusion that saponins are not the only autotoxic agents
in alfalfa (Miller, 1983). Guenzi et al. (1964) reported that
aqueous extracts of alfalfa contain water-solubleinhibitory
substances that reduce the shoot and root growth of corn
(&a mays L.) seedlings. When reseeding or interseeding
alfalfa to thicken the stand, the incorporation of residues
of old plants into the soil may release water-soluble inhibitory substances that can effect new alfalfa seedlings
(Guenzi et al., 1964). Pathogenic infections do not appear
to be responsible for this inhibition (Klein and Miller, 1980).
More recently, researchers have identified inhibitory
compounds which are involved in alfalfa autotoxicity (Hall
and Henderlong, 1989; Dornbos et al., 1990). A watersoluble phytotoxic substance in alfalfa has been characterized as a phenolic compound (Hall and Henderlong,
1989). Medicarpin, sativan, 4-methoxy-medicarpi11, and
5-methoxysativan in alfalfa foliage have been identified
(Dornbos et al., 1990). Among these compounds, medicarpin was implicated as the allelochemical in alfalfa that
causes autotoxicity. Medicarpin produced by alfalfa and
applied exogenously to alfalfa seeds reduced their germination by 59% after 6 h when used in a filter paper
bioassay (Miller et al., 1988).
Fungicide treatment of seeds (Faix et al., 1979) and weed
control treatments (Eltun et al., 1985) to rejuvenate an
old alfalfa stand or to reestablish alfalfa into a preexisting
stand were usually not successful indicating an allelopathic
reaction. In contrast to these negative results, some researchers have successfully reestablished alfalfa 2 to 3 wk after
an existing stand had been killed. Tesar (1984,1993) compared the reseeding of alfalfa following corn or a fallowed
or nonfallowed alfalfa field and found no difference in alfalfa establishment or evidence of autotoxicity if alfalfa
was seeded 2 wk after plowing an old alfalfa stand or 3
wk after spraying the old stand with glyphosate and no-till
seeding the new stand.
Interest in interplanting, double cropping, no-till planting and nonrotational cropping systems increase the need
to understand allelopathy. Information on the critical concentration of toxic compounds that inhibit seedlings when
reestablishing alfalfa into a former alfalfa field is needed.
There has been no previous work on the potential or differential toxicity of separated alfalfa plant parts to seed germination and growth nor on the lack of water uptake by
a germinating seedling as a mechanism of inhibition.
The present research was conducted (i) to evaluate the
effects of aqueous extract concentration of various alfalfa
plant parts on alfalfa seed germinationand seedling growth;
(ii) to evaluate the allelopathic potential of soil in which
hairy vetch (Vicia villosa Roth) and winter rye (Secale
cereale L.) were the crops preceding alfalfa; and (iii) to
study how aqueous extracts of alfalfa leaves affects water
uptake by alfalfa seeds.
Alfalfa (Medicago sahvu L.) plants contain water-soluble substances
that inhibit the germination and seedling growth of alfalfa. Determining where allelochemicals may be found in alfalfa in the greatest concentration would aid in trying to isolate the compound or compounds
responsible for autotoxicity. This study investigated the allelopathiceffects
of various alfalfa plant parts, and the soil in which alfalfa had been
grown, on alfalfa germination and seedling growth. Aqueous extracts
of alfalfa leaf, stem, flower, seed, and root plant parts were made to
determine their effects on germination and dry weights of hypocotyl,
radicle and total length of 5-d-old alfalfa seedlings over a range of extract concentrations. Soil samples (Flanagan series: fine, montmorillonitic, mesic Aquic Argiudolls) from around alfalfa plants at the vegetative
and reproductive stages were compared with sterilized and nonsterilized
soil formerly seeded with alfalfa, hairy vetch (Vicio villosu Roth), and
winter rye (Secule cereule L.). Increasing the aqueous extracts concentrations of separated alfalfa plant parts significantly inhibited alfalfa
germination, seedling length and weight. Radicle length was more sensitive to extract source than seed germination or hypocotyl length. Based
on 5-d-old alfalfa radicle length growth, and averaged across all extract
concentrations, the degree of toxicity of different alfalfa plant parts and
soil from around alfalfa can be classified in order of decreasing inhibition as follows: leaf, seed, complete plant mixture, soil, root, flower,
and stem. Leaf extracts (I2g kg-') caused a 48% decrease in water u p
take by alfalfa seed. Soil in which alfalfa had previously grown was the
most inhibitory to alfalfa growth after 25 d of growth compared with
soil where winter rye or hairy vetch had previously grown. Inhibitory
effects were greater for soil collected around alfalfa grown at the reproductive than the vegetative growth stage. These data indicate that
alfalfa autotoxicitymay m u l t from a release of one or more water-soluble
compounds from alfalfa leaf tissue.
A
is defined as the direct or indirect harmful or beneficial effects of one plant on another through
the production of chemical compounds that escape into
the environment (Rice, 1984). Autotoxicity is an intraspecificform of allelopathy that occurs when a plant species
releases chemical substances that inhibit or delay germination and growth of the same plant species (Putnam, 1985).
Alfalfa (Medicago sativa L.), a perennial legume forage
crop, contains water-soluble substances that are autotoxic
as well as inhibitory to other species (heterotoxicity).
Researchers have observed the phenomenon whereby
alfalfa shoots and roots release inhibitory compounds in
field, greenhouse, and laboratory studies (Nielsen et al.,
1960; Klein and Miller, 1980; Jensen et al., 1984; Tesar,
1984; Hegde and Miller, 1990). Although several compounds have been studied, none have been conclusively
identified as the main autotoxic factor. Li (1981) reported
that alfalfa root aqueous extracts reduced alfalfa seed germination and root development when the extract was inLLELOPATHY
Ill-Min Chung, Dep. of Agronomy, Kon-Kuk Univ., Seoul, South Korea;
and D.A. Miller, Dep. of Agronomy, Univ. of Illinois, Urbana-Champaign,
IL 61801. Exp. Stn. Project 1-6-55179. Received 13 May 1994. *Corresponding author (Email: [email protected]).
Putilished in Agron. J. 87:762-767 (1995).
762
CHUNG & MILLER: ALFALFA AUTOTOXICITY, GERMINATION, AND GROWTH
MATERIALS AND METHODS
Plant and Soil Sampling, Plus Preparation of Extracts
Entire 2-yr-old alfalfa plants (cv. Vernal) grown at the Agronomy and Plant Pathology South Farm, University of Illinois,
were harvested at the vegetative and full bloom stage in August
1991. Fresh alfalfa plants were separated into leaves, stems, roots,
and flowers for each stage. Samples of the Flanagan soil series
(fine, montmorillonitic, mesic Aquic Argiudolls) were collected
from a depth of 18 cm from the area around alfalfa plants at
the vegetative (height 10 cm) and reproductive (flower) growth
stages, respectively. Fresh tissue from each plant part, as well
as soil, was soaked in distilled water for 24 h at 24°C in a lighted
room to give concentrations of 3, 6, 9, and 12 g of tissue or soil
per 100 mL of water. These solutions were filtered through four
layers of cheese cloth to remove the fiber debris and centrifuged
at a low speed (3000 revolutions min-') for 4 h. The supernatant was filtered through filter paper (Whatman no. 42). The
solutions were filtered again using a 0.2-mm Nalgene filterware
unit (Becton Dickinson Labware, Lincoln Park, NJ). Tenmilliliter aliquots from each plant part extract were mixed together to evaluate whole-plant extracts.
Seed Bioassay
Germination tests were conducted for ehch of the respective
plant part extracts as follows: 100 alfalfa seeds (cv. WL-320)
were surface-sterilized with a 1O:l watedbleach (5.25% w/v
NaOCI) solution and were evenly placed on filter paper (Whatman no. 1) in sterilized 9-cm petri dishes. Ten milliliters of extract solution from each plant part were added to each petri dish
and distilled water was used as a control. All petri dishes were
placed in a lighted room at 24°C. Treatments were arranged
in a completely randomized design with four replications. Germination was determined by counting the number of germinated
seeds at 24 h intervals over a 4-d period and expressed as total
percent germinated. Radicle and hypocotyl lengths were determined 5 d after seeding by measuring 24 representative seedlings. After measuring the radicle and hypocotyl lengths, the
seedlings were separated into cotyledon, hypocotyl, and radicle
parts for measuring dry weight.
Water Uptake
One-gram samples of alfalfa seeds were soaked for 8, 16,24,
and 48 h in alfalfa leaf aqueous extracts of 3, 6, 9, and 12 g
100 mL-' water. After an 8-h interval, seeds were taken from
the solution, blotted for 2 h between two folds of filter paper
(Whatman no. 42), and weighed. The water uptake was calculated by subtracting the original seed weight from the final seed
weight. Distilled water was used as the control.
Greenhouse Experiment
A greenhouse experiment was conducted to determine if soil
in which hairy vetch and winter rye had been grown has potential for an allelopathic effect on alfalfa growth. Soil samples (Cisne
silt loam series: fine, montmorillonitic, mesic Mollic Albaqualfs)
were collected to a depth of 18 cm from a 4-yr-old alfalfa stand
at Brownstown Agronomy Research Center in August of 1991,
during the vegetative and reproductive growth stages of alfalfa.
Soil was collected around hairy vetch when it was in the vegetative and reproductive growth stages, and around winter rye
when it was in the headed stage. Each soil sample was air-dried
for 2 mo in the greenhouse and ground to pass through a 2.0-mesh
screen. Soil samples from both stages of alfalfa growth were
divided into two groups. One group was steam-sterilized for 5 h
and the second group was untreated. Each soil sample (500 g)
763
was placed in 11-cm clay pots, and 50 alfalfa seeds (cv. WL-320)
were planted in each pot. Pots were not thinned following seeding.
All pots were steam-sterilized and seeds were surface-sterilized
before use. A sponge plug was placed at the bottom of each pot
to prevent soil loss through the holes in the bottom. A plastic
saucer was placed under each pot to prevent loss of water-soluble
phytotoxic compounds from the pot. Hoagland solution I (Hoagland and Amon, 1950) was applied to the pot saucer as needed
to maintain water status and provide mineral nutrients for alfalfa
growth. All pots were placed in a greenhouse at 24°C in a cycle
of 16 h illumination and 8 h darkness. Light was supplemented
with incandescent and fluorescent lamps. The photosynthetic
photon flux density (PPFD) during illumination was 530 mmol
photon m-2 s-I.
A sterilized soil-sand-peat mixture (1:l:l by volume) and silica sand were used as controls. The treatments and two controls
were arranged in a completely randomized design with four replications. Plant height and dry weights of the shoot and leaves
were measured 25 days after planting.
Statistical Analysis
Analysis of variance for all data was accomplished using the
general linear model procedure of the statistical analysis system
program (SAS Inst., 1985). The experiments were repeated twice
and the pooled mean values were separated on the basis of least
significant difference (LSD) at the 0.05 probability level. Since
there was no significantdifference between the two types of soil
controls in the greenhouse study, these results were combined
and the average used as a control for comparison.
RESULTS AND DISCUSSION
Germination Percentage
Extracts from fresh alfalfa plant leaves, stems, flowers,
seeds, root, soil, and mixture solutions showed inhibitory
effects on seed germination, seedling growth, and seedling weight. The degree of inhibition increased with the
extract concentration. At the highest extract concentration
(12 g kg-'), all aqueous extracts significantly reduced
seed germination compared with the distilled water control (Table 1). Flower extracts were the most inhibitory
at all concentrations while the extract of the mixture of
all plant parts was the least inhibitory. The degree of reduction increased as the extract concentration progressively
increased from 3 to 12 g kg-'. The effect of leaf extracts
were statistically similar to those of flower extracts at the
3 g kg-' concentration. Seed extract reduced germination
to 39% at the 12 g kg-I concentration. Combining all individual extracts had no significant effect on germination
except at the 12 g kg-l concentration. These results are
similar to those of Ballester et al. (1979), who reported
that the most inhibitory effect of Erica plants was produced
by flower extracts.
Hypocotyl Length
Hypocotyl length was not affected by the soil extracts at
3 and 6 g kg-' and even increased 9% at the 3 g kg-l
concentration but all other extracts caused a reduction (Table
2). At the 3 g kg-' concentration, the flower extract
caused the greatest reduction in hypocotyl length (28%)
when compared with other part extracts. The mixture of
all the extracts significantly reduced hypocotyl length at
all concentrations when compared with the control.
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AGRONOMY JOURNAL, VOL. 87, JULY-AUGUST 1995
Table 1. Influence of various concentrations of different aqueous
extracts made from alfalfa plant parts or soil on the germination
of alfalfa seed.?
Table 3. Influence of various concentrations of different aqueous
extracts made from alfalfa plant parts or soil on the radicle length
of 5-d-old alfalfa seedlings.?
~~
Germination. bv extract conc..
g kg-l
Extractant
12
LSD
(0.05)
52.8
67.5
28.0
69.0
59.8
67.0
91.0
48.3
61.0
19.3
39.0
47.8
56.0
81.0
8.4
8.6
6.2
9.3
18.0
1.2
9.8
9.7
6.1
3
6
9
71.5
84.3
61.8
88.3
92.0
85.0
93.0
65.8
72.0
38.8
80.5
65.3
74.0
91.0
10.3
9.7
Radicle length, by extract
conc., g kg-'
Extractant
3
6
9
2.4
3.1
2.9
2.6
3.8
2.5
2.7
2.2
3.3
2.8
2.4
2.8
2.6
2.6
cm
1 .o
3.0
2.5
1.3
1.5
2.4
1.7
0.2
0.2
0.2
%
Leaf
Stem
Flower
Seed
Root
Soil
Mixture
Control = 93.5%
LSD (0.05)
t Leaf, stem, and root extracts, obtained from vegetative plants; flower and
seed extracts obtained from reproductive plants. For soil extracts, the soil
in the immediate vicinity of a vegetative alfalfa plant was used. The mixture was prepared by mixing equal parts from leaf, stem, flower, seed, and
root extracts.
Leaf
Stem
Flower
Seed
Root
Soil
Mixture
Control = 4.5 cm
LSD (0.05)
12
0.8
2.6
2.4
1 .o
1.0
1.8
1.0
0.3
LSD
(0.05)
0.2
0.3
0.1
0.1
0.1
0.6
0.2
~~_____
t Leaf, stem, and root extracts, obtained from vegetative plants; flower and
seed extracts obtained from reproductive plants. For soil extracts, the soil
in the immediate vicinity of a vegetative alfalfa plant was used. The mixture was prepared by mixing equal parts from leaf, stem, flower, seed, and
root extracts.
Radicle Length
Total Seedling Length
Radicle length was relatively more sensitive to autotoxic
allelochemicals than was hypocotyl length, except for stem
and flower extracts. All extracts caused a marked reduction in radicle length of alfalfa seedlings (Table 3). An
especially high degree of inhibition occurred with leaf extracts except at 12 g kg-' concentration. Seed extracts also
exhibited a pronounced reduction in radicle elongation.
Besides inhibiting radicle elongation, other morphological abnormalities occurred as many of the extracts caused
twisted radicle growth. The most severely twisted roots
were observed in seedlings treated with leaf extracts. Hall
and Henderlong (1989) also found that seedling elongation of alfalfa was inhibited more by alfalfa autotoxic compounds than was germination. Based on significant radicle length reactions to aqueous extracts, averaged over all
extract concentrations, and seedling weight, the toxicity
may be classified in the following order of decreasing inhibition: leaf, seed, mixture of parts, root, soil, flowers,
and stem.
Total seedling length has been viewed as being generally more sensitive to inhibitory compounds than seed germination and shoot elongation (Hall and Henderlong, 1989;
Kuiters, 1989; Luu et al., 1989; Hegde and Miller, 1990).
Thus, overall seedling growth may be the best indicator
of sensitivity to allelochemicals (Rietveld, 1983). Total
seedling length was more inhibited by the leaf extracts
at 3 g kg-' than was germination percentage: 35 and
24%, respectively (Tables 1 and 4). At the 3 g kg-' concentration, flower extracts inhibited the total seedling length
by 34%. Comparing all other extractants at the 3 g kg-'
concentration, total seedling length was more inhibited
than germination percentage and in descending order:
respectively, seed, 31 vs. 6%; mixture, 27 vs. 1%; soil,
22 vs. 9%; root, 14 vs. 2%; and stem, 14 vs. 10%.
In this study, aqueous soil extracts significantly inhibited alfalfa seed germination and seedling growth when
Table 2. Influence of various concentrations of different aqueous
extracts made from alfalfa plant parts or soil on the hypocotyl
length of 5-d-old alfalfa seedlings.?
Table 4. Influence of various concentrations of different aqueous
extracts made from alfalfa plant parts or soil on the total length
of 5-d-old alfalfa seedlings.?
Hypocotyl length,
by extract conc., g kg-'
Extractant
Leaf
Stem
Flower
Seed
Root
Soil
Mixture
Control = 3.2 cm
LSD (0.05)
3
6
9
2.6
2.9
2.2
2.8
2.8
2.9
2.6
2.4
2.2
2.7
2.1
3.0
2.9
cm
2.2
2.6
2.1
2.4
1.9
2.6
2.1
2.5
2.0
0.2
0.2
0.3
0.3
3.5
12
1.8
2.4
2.0
2.0
1.3
LSD
(0.05)
0.3
0.3
0.1
0.1
0.1
0.5
0.2
+ Leaf, stem, and root extracts, obtained from vegetative plants; flower and
seed extracts obtained from reproductive plants. For soil extracts, the soil
in the immediate vicinity of a vegetative alfalfa plant was used. The mixture was prepared by mixing equal parts from leaf, stem, flower, seed, root,
and soil extracts.
Seedling Weight
Total length, by extract conc.,
g kg-'
Extractant
Leaf
Stem
Flower
Seed
Root
Soil
Mixture
Control = 7.7 cm
LSD (0.05)
3
6
9
12
LSD
(0.05)
5.0
6.6
5.1
5.3
6.6
6.0
5.6
4.7
5.7
4.9
5.1
4.9
5.5
5.5
cm
3.2
5.6
4.7
3.7
3.4
5.0
3.8
2.6
4.9
4.4
3.0
2.2
4.3
3.0
0.4
0.5
0.1
0.1
0.3
0.8
0.3
0.4
0.4
0.3
0.5
t Leaf, stem, and root extracts, obtained from vegetative plants; flower and
seed extracts obtained from reproductive plants. For soil extracts, the soil
in the immediate vicinity of a vegetative alfalfa plant was used. The mixture was prepared by mixing equal parts from leaf, stem, flower, seed, and
root extracts.
765
CHUNG & MILLER: ALFALFA AUTOTOXICITY, GERMINATION, AND GROWTH
Table 5. Influence of various concentrations of different aqueous
extracts made from alfalfa plant parts or soil on the dry weight
of cotyledons of 5-d-old alfalfa seedlings.?
Cotyledon dry wt., by extract
conc., g kg-'
Extractant
Leaf
Stem
Flower
Seed
Root
Soil
Mixture
Control = 1.00 mg
LSD (0.05)
3
6
9
0.93
0.95
1.03
0.98
0.83
0.83
0.95
0.88
0.95
0.95
0.90
0.70
0.78
0.90
mg
0.80
0.85
0.78
0.83
0.80
0.73
0.85
0.08
0.06
0.08
12
LSD
(0.05)
0.75
0.75
0.65
0.75
0.68
0.70
0.78
0.07
0.08
0.08
0.07
0.08
0.09
0.07
Table 7. Influence of various concentrations of different aqueous
extracts made from alfalfa plant parts or soil on the dry weight
of the radicle of 5-d-old alfalfa seedlings.?
Radicle dry wt., by extract
conc., g kg-'
Extractant
3
6
9
12
LSD
(0.05)
0.43
0.40
0.28
0.35
0.30
0.33
0.43
0.43
0.40
0.25
0.30
0.30
0.28
0.30
mg
0.30
0.30
0.18
0.23
0.23
0.25
0.20
0.35
0.25
0.15
0.23
0.20
0.23
0.18
0.07
0.04
0.08
0.07
0.03
0.11
0.08
0.06
0.07
~
0.09
Leaf
Stem
Flower
Seed
Root
Soil
Mixture
Control = 0.53
LSD (0.05)
0.07
~
0.07
~
t Leaf, stem, and root extracts, obtained from vegetative plants; flower and
t Leaf, stem, and root extracts, obtained from vegetative plants; flower and
seed extracts obtained from reproductive plants. For soil extracts, the soil
in the immediate vicinity of a vegetative alfalfa plant was used. The mixture was prepared by mixing equal parts from leaf, stem, flower, seed, and
root extracts.
seed extracts obtained from reproductive plants. For soil extracts, the soil
in the immediate vicinity of a vegetative alfalfa plant was used. The mixture was prepared by mixing equal parts from leaf, stem, flower, seed, and
root extracts.
compared with distilled water control (Tables 1 and 4).
The root and soil extracts reduced cotyledon dry weight
significantly more than extracts from most other plant parts
at the 3 and 6 g kg-' extract concentrations (Table 5 ) .
These results are in contrast to those of Jensen et al. (1984),
who found that soil extracts did not significantly inhibit
seed germination and seedling growth. Compared with
the control, hypocotyl dry weight (Table 6) was significantly inhibited by all extract concentrations. The flower
and seed extracts at the 3 g kg-' concentration reduced
hypocotyl dry weight by 29% and 36%,respectively while
the leaf and flower extracts at the 12 g kg-' concentration
reduced hypocotyl dry weight by 63%. Stem and flower
extracts at the 6 g kg-' concentration reduced hypocotyl
dry weight by 43% and 49% when compared with the control. Radicle dry weight tended to decrease as the extract
concentration increased (Table 7). The root extract was
the most inhibitory at the 3 g kg-' concentration and reduced total dry weight of seedlings by 24%, while root
and soil extracts at the 6 g kg-' concentration reduced
total seedling dry weight by 30% (Table 8).
Table 6. Influence of various concentrations of different aqueous
extracts made from alfalfa plant parts or soil on the dry weight
of the hypocotyl of 5-d-old alfalfa seedlings.?
Hypocotyl dry wt., by extract
conc., g kg-'
Extractant
3
6
9
12
LSD
(0.05)
Water Uptake
Many enzymatic functions important to plants are inhibited by the presence of allelochemicals (Rice, 1984).
In high protein seeds like alfalfa, proteases play an important role in the hydrolysis of proteins during germination. To a large extent the activity of these enzymes is
primarily related to water imbibition by the seeds. Although
enzyme activity was not investigated in this study, an indirect association between lower seed germination and
allelopathic inhibition may be the consequence of the inhibition of water uptake and enzyme activity (Table 9).
Increasing the concentration of aqueous leaf extracts significantly inhibited the water uptake by germinating alfalfa
seeds. The greatest inhibition in water uptake occurred
at the 12 g kg-' extract concentration for seeds soaked
for 8 h. After 24 h, there was a decrease in water uptake
(Table 9).
Greenhouse Experiment
Compared with the control, some aspects of alfalfa
growth (plant height, leaf area, dry leaf, and stem weight)
Table 8. Influence of various concentrations of different aqueous
extracts made from alfalfa plant parts or soil on the total dry weight
of 5-d-old alfalfa seedlings.?
Total dry wt., by extract
conc., g kg-'
Extractant
3
6
9
12
LSD
(0.05)
1.98
1.95
1.78
1.85
1.73
1.83
2.05
1.80
1.78
1.58
1.70
1.60
1.60
1.75
mg
1.55
1.55
1.30
1.48
1.63
1.53
1.40
1.38
1.40
1.05
1.35
1.30
1.43
1.30
0.11
0.11
0.21
0.07
0.10
0.14
0.17
0.11
0.11
0.12
0.15
~~
Leaf
Stem
Flower
Seed
Root
Soil
Mixture
Control = 0.75 mg
LSD (0.05)
0.63
0.60
0.48
0.53
0.60
0.68
0.68
0.50
0.43
0.38
0.50
0.60
0.55
0.55
mg
0.45
0.40
0.35
0.43
0.60
0.55
0.35
0.28
0.40
0.25
0.38
0.43
0.50
0.35
0.07
0.06
0.07
0.08
0.07
0.03
0.08
0.07
0.03
0.09
0.10
Leaf
Stem
Flower
Seed
Root
Soil
Mixture
Control = 2.28 mg
LSD (0.05)
t Leaf, stem, and root extracts, obtained from vegetative plants; flower and
t Leaf, stem, and root extracts, obtained from vegetative plants; flower and
seed extracts obtained from reproductive plants. For soil extracts, the soil
in the immediate vicinity of a vegetative alfalfa plant was used. The mixture was prepared by mixing equal parts from leaf, stem, flower, seed, and
root extracts.
seed extracts obtained from reproductive plants. For soil extracts, the soil
in the immediate vicinity of a vegetative alfalfa plant was used. The mixture was prepared by mixing equal parts from leaf, stem, flower, seed, and
root extracts.
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AGRONOMY JOURNAL, VOL. 87, JULY-AUGUST 1995
Table 9. Total water uptake by alfalfa seeds treated with different
concentration of aqueous extracts of vegetative stage leaves at
different soaking periods.
Water uptake, by soaking time (h)
Concentration
16
24
48
(0.05)
1.04
0.68
0.64
0.60
0.54
0.02
1.05
0.91
0.82
0.80
0.69
0.06
——— g ——
1.19
0.97
0.94
0.93
0.89
0.03
1.09
0.93
0.86
0.79
0.71
0.04
0.03
0.04
0.02
0.03
0.07
gkg-'
0.0
3.0
6.0
9.0
12.0
LSD (0.05)
LSD
8
were significantly inhibited in soil previously planted with
winter rye, hairy vetch, and alfalfa (Table 10). Miller (1983)
reported that reseeding alfalfa into areas of an old alfalfa
field may result in erratic stands. Our results suggest that
autotoxic compounds can accumulate to sufficiently high
concentrations to be bioactive. These allelochemicals may
have been released through leaching and decomposition
of alfalfa residue. Some evidence for this view is indicated
by the 0.9 to 0.7 unit reduction in pH for alfalfa soil when
compared with the control (Table 10). Alfalfa plant height
was not significantly different for winter rye and hairy
vetch soil, but leaf area was significantly reduced when
the preceding crop was hairy vetch (Table 10).
The influence of soil sampled from around alfalfa varied with growth stages in terms of alfalfa autotoxicity. A
comparison of soil pH values from the vegetative and reproductive stage samples (Table 10) implies that there may
be some chemical release influencing the pH between the
two growth stages and the control soil. Nonsterilized soil
collected from the reproductive alfalfa stage was more inhibitory to plant height and leaf area than soil from the
vegetative stage. This observation suggests that autotoxic
compounds may accumulate in the soil as plant growth
progresses. No difference was observed in autotoxicity
between sterilized and nonsterilized soil at the vegetative
alfalfa stage. Nonsterilized soil collected during reproductive stage of alfalfa was more inhibitory to plant height
than sterilized soil. Such a response could indicate that
microbial activity may be involved in the release of phytotoxic substances that directly affect growth.
Researchers have reported that winter rye can exhibit
allelopathic responses when used as a cover crop (Liebl
and Worsham, 1983). Similarly, there is some indication
that hairy vetch residue may help in weed suppression
Table 10. Influence of various soil treatments on growth of alfalfa
following 25 d of growth and on the pH of each soil.
Treatment
Control soil
Winter rye soil
Hairy vetch soil
Vegetative alfalfa soil
(VAS), sterilized
VAS, nonsterilized
Reproductive alfalfa soil
(RAS), sterilized
RAS, nonsterilized
LSD (0.05)
Plant
height
Leaf
area
2
Dry wt.
Stem
Leaf
PH
——— mg ———
14.6
13.1
12.8
11.4
12.4
10.1
11.0
10.6
6.9
6.8
6.5
6.2
6.0
cm
20.1
17.4
17.2
15.0
cm
5.2
4.9
4.5
15.1
15.3
4.4
10.9
10.7
10.7
3.9
13.5
3.7
0.4
10.4
1.9
9.4
1.8
0.9
4.4
9.7
-
(Teasdale, 1988). The reduction in alfalfa seedling growth
from the hairy vetch soil treatment in this study (Table 10)
supports the view that vetch may add allelochemicals to
the soil that persist well into the growing season.
It is difficult to apply our results to a production situation directly, because the concentration of inhibitory substances in aqueous extracts is probably greater than what
would be observed under natural conditions. Depending
on the amount of alfalfa residue and the stage of growth
when tillage occurs, allelopathic activity will vary when
reseeding alfalfa. Clearly, the role of microbial activity
and allelopathic potential of a preceding hairy vetch soil
needs further study. Further investigations are also needed
to determine the influence of seasonal and cultivar variations, and to identify the active compounds involved in
alfalfa autotoxicity and allelopathy.
CHUNG & MILLER: ALFALFA AUTOTOXICITY, GERMINATION, AND GROWTH
767